Novel antibody conjugates reactive with human carcinomas

ABSTRACT

The present invention relates to novel antibodies, antibody fragments and antibody conjugates and single-chain immunotoxins reactive with human carcinoma cells. More particularly, the antibodies, conjugates and single-chain immunotoxins of the invention include: a murine monoclonal antibody, BR96; a human/murine chimeric antibody, ChiBR96; a F(ab′) 2  fragment of BR96; ChiBR96-PE, ChiBR96-LysPE40, ChiBR96 F(ab′) 2 -LysPE40 and ChiBR96 Fab′-LysPE40 conjugates and recombinant BR96 sFv-PE40 immunotoxin. These molecules are reactive with a cell membrane antigen on the surface of human carcinomas. The BR96 antibody and its functional equivalents, displays a high degree of selectivity for carcinoma cells and possess the ability to mediate antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. In addition, the antibodies of the invention internalize within the carcinoma cells to which they bind and are therefore particularly useful for therapeutic applications, for example, as the antibody component of antibody-drug or antibody-toxin conjugates. The antibodies also have a unique feature in that they are cytotoxic when used in the unmodified form, at specified concentrations.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.08/057,444, filed May 5, 1993, which is a file wrapper continuationapplication of U.S. Ser. No. 07/544,246 filed Jun. 26, 1990, which was acontinuation-in-part of U.S. Ser. No. 07/374,947, filed Jun. 30, 1989,now abandoned, the entire disclosure of these applications beingincorporated by reference herein.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to novel antibodies reactive withcarcinoma cells. More particularly, the invention relates to a murinemonoclonal antibody and a chimeric monoclonal antibody, includingimmunoconjugates and recombinant immunotoxins made therefrom, that reactwith cell membrane antigens associated with a large variety ofcarcinomas including carcinomas of the colon, breast, ovary and lung.The murine monoclonal antibody is highly specific for carcinomas,showing no to very low reactivity with normal animal tissues or othertypes of tumors such as lymphomas or sarcomas.

BACKGROUND OF THE INVENTION

[0003] 1. Monoclonal Antibodies Directed Against Cell Membrane Antigens

[0004] Monoclonal antibodies (MAbs) to human tumor-associateddifferentiation antigens offer promises for the “targeting” of variousantitumor agents such as radioisotopes, chemotherapeutic drugs, andtoxins. (Order, in “Monoclonal Antibodies for Cancer Detection andTherapy,” Baldwin and Byers, (eds.), London, Academic Press (1985)).

[0005] In addition, some monoclonal antibodies have the advantage ofkilling tumor cells via antibody-dependent cellular cytotoxicity (ADCC)or complement-dependent cytotoxicity (CDC) in the presence of humaneffector cells or serum (Hellstrom et al., Proc. Natl. Acad. Sci. USA83:7059-7063 (1986)), and there are a few monoclonal antibodies thathave a direct antitumor activity which does not depend on any hostcomponent (Drebin et al., Oncogene 2:387-394 (1988)).

[0006] Many monoclonal antibodies reactive with carcinoma-associatedantigens are known (see, e.g., Papsidero, “Recent Progress In TheImmunological Monitoring of Carcinomas Using Monoclonal Antibodies,Semin. Surg. Oncol. 1(4):171-81 (1985); Schlom et al., “PotentialClinical Utility Of Monoclonal Antibodies In The Management Of HumanCarcinomas,” Important Adv. Oncol., 170-92 (1985); Allum et al.,“Monoclonal Antibodies In The Diagnosis And Treatment of MalignantConditions,” Surg. Ann. 18:41-64 (1986); and Houghton et al.,“Monoclonal Antibodies: Potential Applications To The Treatment OfCancer,” Semin. Oncol., 13(2):165-79 (1986)).

[0007] These known monoclonal antibodies can bind to a variety ofdifferent carcinoma-associated antigens including glycoproteins,glycolipids and mucins (see, e.g., Fink et al., “Monoclonal AntibodiesAs Diagnostic Reagents for The Identification And Characterization OfHuman Tumor Antigens,” Prog. Clin. Pathol., 9:121-33 (1984)).

[0008] For example, monoclonal antibodies that bind to glycoproteinantigens on specific types of carcinomas include those described in U.S.Pat. No. 4,737,579 (monoclonal antibodies to non-small cell lungcarcinomas), U.S. Pat. No. 4,753,894 (monoclonal antibodies to humanbreast cancer), U.S. Pat. No. 4,579,827 (monoclonal antibodies to humangastrointestinal cancer), and U.S. Pat. No. 4,713,352 (monoclonalantibodies to human renal carcinoma).

[0009] Monoclonal antibody B72.3, which is one of the antibodies studiedthe most, recognizes a tumor-associated mucin antigen of greater than1,000 kd molecular weight that is selectively expressed on a number ofdifferent carcinomas. Thus, B72.3 has been shown to react with 84% ofbreast carcinomas, 94% of colon carcinomas, 100% of ovarian carcinomasand 96% of non-small cell lung carcinomas (see Johnston, “Applicationsof Monoclonal Antibodies In Clinical Cytology As Exemplified By StudiesWith Monoclonal Antibody B72.3,” Acta Cytol. 1(5):537-56 (1987) and U.S.Pat. No. 4,612,282, issued to Schlom et al.). Another patentedmonoclonal antibody, KC-4, (see U.S. Pat. No. 4,708,930), recognizes anapproximately 400-500 kd protein antigen expressed on a number ofcarcinomas, such as colon, prostate, lung and breast carcinoma. Itappears that neither the B72.3 nor KC-4 antibodies internalize withinthe carcinoma cells with which they react.

[0010] Monoclonal antibodies reactive with glycolipid antigensassociated with tumor cells have been disclosed. For example, Young etal., “Production Of Monoclonal Antibodies Specific For Two DistinctSteric Portions of The Glycolipid Ganglio-N-Triosylceramide (AsialoGM₂).” J. Exp. Med., 150:1008-1019 (1979) disclose the production of twomonoclonal antibodies specific for asialo GM₂, a cell surfaceglycosphingolipid antigen that was established as a marker for BALB/cV3T3 cells transformed by Kirsten murine sarcoma virus. See, also, Kniepet al., “Gangliotriasylceramide (Asialo GM₂) A Glycosphingolipid MarkerFor Cell Lines Derived From Patients With Hodgkin's Disease,” J.Immunol. 131(3):1591-94 (1983) and U.S. Pat. No. 4,507,391 (monoclonalantibody to human melanoma).

[0011] Other monoclonal antibodies reactive with glycolipid antigens oncarcinoma cells include those described by Rosen et al., “Analysis OfHuman Small Cell Lung Cancer Differentiation Antigens Using A Panel OfRat Monoclonal Antibodies,” Cancer Research, 44:2052-61 (1984)(monoclonal antibodies to human small cell lung cancer), Varki et al.,“Antigens Associated with a Human Lung Adenocarcinoma Defined byMonoclonal Antibodies,” Cancer Research 44:681-87 (1984); (monoclonalantibodies to human adenocarcinomas of the lung, stomach and colon andmelanoma), and U.S. Pat. No. 4,579,827 (monoclonal antibodies to humancolon adenocarcinoma). See, also, Hellstrom et al., “Antitumor EffectsOf L6, An IgG2a Antibody That Reacts With Most Human Carcinomas,” Proc.Natl. Acad. Sci. USA, 83:7059-63 (1986) which describes the L6monoclonal antibody that recognizes a carbohydrate antigen expressed onthe surface of human non-small cell lung carcinomas, breast carcinomasand colon carcinomas.

[0012] Antibodies to tumor-associated antigens which are not able tointernalize within the tumor cells to which they bind are generally notuseful to prepare conjugates with antitumor drugs or toxins, since thesewould not be able to reach their site of action within the cell. Otherapproaches would then be needed so as to use such antibodiestherapeutically.

[0013] Additional monoclonal antibodies exhibiting a high specificreactivity to the majority of cells from a wide range of carcinomas aregreatly needed. This is so because of the antigenic heterogeneity ofmany carcinomas which often necessitates, in diagnosis or therapy, theuse of a number of different monoclonal antibodies to the same tumormass. There is a further need, especially for therapy, for so called“internalizing” antibodies, i.e., antibodies that are easily taken up bythe tumor cells to which they bind. Antibodies of this type find use intherapeutic methods for selective cell killing utilizing antibody-drugor antibody-toxin conjugates (“immunotoxins”) wherein a therapeuticantitumor agent is chemically or biologically linked to an antibody orgrowth factor for delivery to the tumor, where the antibody binds to thetumor-associated antigen or receptor with which it is reactive and“delivers” the antitumor agent inside the tumor cells (see, e.g.,Embleton et al., “Antibody Targeting Of Anti-Cancer Agents,” inMonoclonal Antibodies For Cancer Detection and Therapy, pp. 317-44(Academic Press, 1985)).

[0014] 2. Immunotoxins

[0015] Immunotoxins have been investigated as a new approach fortreating metastatic tumors in man (Pastan and FitzGerald, Science254:1173-1177 (1991); FitzGerald and Pastan, Seminars in Cell Biology2:31-37 (1991) and Vitetta et al., Science 644:650 (1987)). Pseudomonasexotoxin A (“PE”) is a cytotoxic agent produced by Pseudomonasaeruginosa that kills cells by ADP-ribosylating elongation factor 2,thereby inhibiting protein synthesis (Iglewski et al., Proc. Natl. Acad.Sci. USA 72:2284-2285 (1975)).

[0016] PE is a polypeptide comprising three domains (Allured et al.,Proc. Natl. Acad. Sci. USA 83:1320-1324 (1986)).

[0017] Domain I encodes the cell-binding ability; domain II encodes theproteolytic sensitivity site and the membrane translocation ability; anddomain III encodes the ADP-ribosylation activity of the toxin (Hwang etal., Cell 48:129-136 (1987), Siegall et al., J. Biol. Chem.264:14256-14261 (1989)). By removing domain I from PE, a truncated 40kDa toxin is formed (“PE40”) (Kondo et al., J. Biol. Chem. 263:9470-9475(1988)).

[0018] PE40 is weakly toxic to cells because it lacks the cell bindingdomain for the PE receptor (Id.) For conjugation of this molecule to anantibody, the amino terminus of PE40 is modified to include a lysineresidue to form “LysPE40” (Batra et al., supra). Immunotoxins using PE,have shown promise in preclinical models using human tumor xenografts innude mice (Batra et al., Proc. Natl. Acad. Sci. USA 86:8545-8549 (1989);and Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362 (1991)).

[0019] Several internalizing antibodies reacting with lymphocyteantigens are known. In contrast, such antibodies are rare when dealingwith solid tumors. One of the few examples of an internalizing antibodyreacting with carcinomas is an antibody disclosed in Domingo et al.,“Transferrin Receptor As A Target For Antibody-Drug Conjugates,” MethodsEnzymol. 112:238-47 (1985). This antibody is reactive with the humantransferrin-receptor glycoprotein expressed on tumor cells. However,because the transferrin-receptor is also expressed on many normaltissues, and often at high levels, the use of ananti-transferrin-receptor antibody in an antibody-drug or antibody-toxinconjugate may have significant toxic effects on normal cells. Theutility of this antibody for selective killing or inhibition of tumorcells is therefore questionable. Another internalizing antibody is BR64(disclosed in co-pending patent applications U.S. Ser. No. 289,635,filed Dec. 22, 1988, and Ser. No. 443,696 filed Nov. 29, 1989, andincorporated by reference herein), which binds to a large spectrum ofhuman carcinomas.

[0020] 3. Chimeric Antibodies

[0021] The cell fusion technique for the production of monoclonalantibodies (Kohler and Milstein, Nature (London) 256:495 (1975)) haspermitted the development of a number of murine monoclonal antibodiesreactive with antigens, including previously unknown antigens.

[0022] However, murine monoclonal antibodies may be recognized asforeign substances by the human immune system and neutralized such thattheir potential in human therapy is not realized. Therefore, recentefforts have focused on the production of so-called “chimeric”antibodies by the introduction of DNA into mammalian cells to obtainexpression of immunoglobulin genes (Oi et al., Proc. Natl. Acad. Sci.USA 80:825 (1983); Potter et al., Proc. Natl. Acad. Sci. USA 81:7161;Morrison et al., Proc. Natl. Acad. Sci. USA 81:6581 (1984); Sahagan etal., J. Immunol. 137:1066 (1986); Sun et al., Proc. Natl. Acad. Sci.84:214 (1987)).

[0023] Chimeric antibodies are immunoglobulin molecules comprising ahuman and non-human portion. More specifically, the antigen combiningregion (variable region) of a chimeric antibody is derived from anon-human source (e.g., murine) and the constant region of the chimericantibody which confers biological effector function to theimmunoglobulin is derived from a human source. The chimeric antibodyshould have the antigen binding specificity of the non-human antibodymolecule and the effector function conferred by the human antibodymolecule.

[0024] In general, the procedures used to produce chimeric antibodiesinvolve the following steps:

[0025] a) identifying and cloning the correct gene segment encoding theantigen binding portion of the antibody molecule; this gene segment(known as the VDJ, variable, diversity and joining regions for heavychains or VJ, variable, joining regions for light chains or simply asthe V or variable region) may be in either the cDNA or genomic form;

[0026] b) cloning the gene segments encoding the constant region ordesired part thereof;

[0027] c) ligating the variable region with the constant region so thatthe complete chimeric antibody is encoded in a form that can betranscribed and translated;

[0028] d) ligating this construct into a vector containing a selectablemarker and gene control regions such as promoters, enhancers and poly(A) addition signals;

[0029] e) amplifying this construct in bacteria;

[0030] f) introducing this DNA into eukaryotic cells (transfection) mostoften mammalian lymphocytes;

[0031] g) selecting for cells expressing the selectable marker;

[0032] h) screening for cells expressing the desired chimeric antibody;and

[0033] i) testing the antibody for appropriate binding specificity andeffector functions.

[0034] Antibodies of several distinct antigen binding specificities havebeen manipulated by these protocols to produce chimeric proteins (e.g.,-anti-TNP: Boulianne et al., Nature 312:643 (1984); and anti-tumorantigens: Sahagan et al., J. Immunol. 137:1066 (1986)). Likewise,several different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseinclude enzymes (Neuberger et al., Nature 312:604 (1984)),immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain (Sharon et al., Nature 309:364(1984); Tan et al., J. Immunol. 135:3565- 3567 (1985)).

[0035] 4. Modifying Genes In Situ Encoding Monoclonal Antibodies

[0036] The discovery of homologous recombination in mammalian cellspermits the targeting of new sequences to specific chromosomal loci.Homologous recombination occurs when cultured mammalian cells integrateexogenous DNA into chromosomal DNA at the chromosome location whichcontains sequences homologous to the plasmid sequences (Folger et al.,Mol. Cell. Biol. 2:1372-1387 (1982); Folger et al., Symp. Quant. Biol.49:123-138 (1984); Kucherlapati et al., Proc. Natl. Acad. Sci. USA81:3153-3157 (1984); Lin et al., Proc. Natl. Acad. Sci. USA 82:1391-1395(1985); de Saint Vincent et al., Proc. Natl. Acad. Sci. USA 80:2002-2006(1983); Shaul et al., Proc. Natl. Acad. Sci. USA 82:3781-3784 (1985)).

[0037] The potential for homologous recombination within cells permitsthe modification of endogenous genes in situ. Conditions have been foundwhere the chromosomal sequence can be modified by introducing into thecell a plasmid DNA which contains a segment of DNA homologous to thetarget locus and a segment of new sequences with the desiredmodification (Thomas et al., Cell 44:419-428 (1986); Smithies et al.,Nature 317:230-234 (1985); Smith et al., Symp. Quant. Biol. 49:171-181(1984)). Homologous recombination between mammalian cell chromosomal DNAand the exogenous plasmid DNA can result in the integration of theplasmid or in the replacement of some of the chromosomal sequences withhomologous plasmid sequences. This can result in placing a desired newsequence at the endogenous target locus.

[0038] The process of homologous recombination has been evaluated usinggenes which offer dominant selection such as NEO and HPRT for a few celltypes (Song et al., Proc. Natl. Acad. Sci. USA 84:6820-6824 (1987);Rubinitz-and Subramani, Mol. Cell Biol. 6:1608-1614 (1986); and Liskay,Cell 35:157-164 (1983)). Recently, procedures for modifying antibodymolecules and for producing chimeric antibody molecules using homologousrecombination to target gene modification have been described (Fell etal., Proc. Natl. Acad. Sci. USA 86:8507-8511 (1989); and co-pending U.S.patent applications Ser. No. 243,873 filed Sep. 14, 1988, and Ser. No.468,035 filed Jan. 22, 1990, assigned to the same assignee as thepresent application, all of which are incorporated by reference herein).

[0039] 5. Monoclonal Antibodies in Therapy

[0040] The most direct way to apply antitumor monoclonal antibodiesclinically is to administer them in unmodified form, using monoclonalantibodies which display antitumor activity in vitro and in animal (suchas humans, dogs, cows, pigs, horses, cats, rats, and mice) models. Mostmonoclonal antibodies to tumor antigens do not appear to have anyantitumor activity by themselves, but certain monoclonal antibodies areknown which mediate complement-dependent cytotoxicity(complement-dependent cytotoxicity), i.e., kill human tumor cells in thepresence of human serum as a source of complement (see, e.g., Hellstromet al., Proc. Natl. Acad. Sci. USA 82:1499-1502 (1985)), orantibody-dependent cellular cytotoxicity (antibody-dependent cellularcytotoxicity) together with effector cells such as human NK cells ormacrophages.

[0041] To detect antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity activity monoclonal antibodies aretested for lysing cultured ⁵¹Cr-labeled tumor target cells over a 4-hourincubation period.

[0042] Target cells are labeled with ⁵¹Cr and then exposed for 4 hoursto a combination of effector cells (in the form of human lymphocytespurified by the use of a lymphocyte-separation medium) and antibody,which is added in concentrations varying between 0.1 μg/ml and 10 μg/ml.The release of ⁵¹Cr from the target cells is measured as evidence oftumor-cell lysis (cytotoxicity). Controls include the incubation oftarget cells alone or with either lymphocytes or monoclonal antibodyseparately.

[0043] The total amount of ⁵¹Cr that can be released is measured andantibody-dependent cellular cytotoxicity is calculated as the percentkilling of target cells observed with monoclonal antibody plus effectorcells as compared to target cells being incubated alone. The procedurefor complement-dependent cytotoxicity is identical to the one used todetect antibody-dependent cellular cytotoxicity except that human serum,as a source of complement, (diluted 1:3 to 1:6) is added in place of theeffector cells.

[0044] Monoclonal antibodies with antibody-dependent cellularcytotoxicity and complement-dependent cytotoxicity activity areconsidered for therapeutic use because they often have anti-tumoractivities in vivo. Antibodies lacking antibody-dependent cellularcytotoxicity and complement-dependent cytotoxicity activity in vitro, onthe other hand, are commonly ineffective in vivo unless used as carriersof antitumor agents.

[0045] The ability of a monoclonal antibody to activate the host'scomplement may prove to be therapeutically beneficial not only becausetumor cells may be killed, but also because the blood supply to tumorsmay increase, thus facilitating the uptake of drugs (see Hellstrom etal., “Immunological Approaches to Tumor Therapy: Monoclonal Antibodies,Tumor Vaccines, and Anti-Idiotypes, in Covalently Modified Antigens andAntibodies in Diagnosis and Therapy, Quash & Rodwell, eds., MarcelDekker, pp. 15-18 (1989)).

[0046] Among mouse monoclonal antibodies, the IgG2a and IgG3 isotypesare most commonly associated with antibody-dependent cellularcytotoxicity and complement-dependent cytotoxicity. Antibodies havingboth antibody-dependent cellular cytotoxicity and complement-dependentcytotoxicity activity have high selectivity for killing only the tumorcells to which they bind and would be unlikely to lead to toxic effectsif non-specifically trapped in lung, liver or other organs. This maygive such antibodies an advantage over radiolabeled antibodies orcertain types of immunoconjugates.

[0047] Therapeutic modalities directed to treating tumors are commonlyavailable. For example, chemotherapy is an effective treatment forselected human tumors. However, with chemotherapy only modest progresshas been made for treating the majority of carcinomas, includingcarcinomas of breast, lung, and colon.

[0048] The introduction of monoclonal antibody (MAb) technology in the1970s raised hopes that tumor-specific MAbs could be used to targetanti-tumor agents and provide more effective therapy (K. E. Hellstrom,and I. Hellstrom, in Biologic Therapy of Cancer: Principles andPractice, V. T. DeVita, S. Hellman, and S. A. Rosenberg, Eds. (J. P.Lippincott Company, Philadelphia, Pa., 1991, pp. 35-52).

[0049] 6. Immunoconjugates in Therapy

[0050] Various immunoconjugates in which antibodies were used to targetchemotherapeutic drugs (P. N. Kulami, A. H. Blair, T. I. Ghose, CancerRes. 41, 2700 (1981); R. Arnon, R. and M. Sela, Immunol. Rev. 62, 5(1982); H. M. Yang and R. A. Resifeld, Proc. Natl. Acad. Sci. U.S.A.,85, 1189 (1988); R. O. Dilman, D. E. Johnson, D. L. Shawler, J. A.Koziol, Cancer Res. 48, 6097 (1988); L. B. Shih, R. M. Sharkey, F. J.Primus, D. M. Goldenberg, Int. J. Cancer 41, 832 (1988); P. A. Trail etal., Cancer Res. 52, 5693 (1992)), or plant and bacterial toxins (I.Pastan, M. C. Willingham, D. J. Fitzgerald, Cell 47, 641 (1986); D. C.Blakey, E. J. Wawrzynczak, P. M. Wallace, P. E. Thorpe, in MonoclonalAntibody Therapy Prog. Allergy, H. Waldmann, Ed. (Karger, Basel, 1988),pp. 50-90) have been evaluated in preclinical models and found to beactive in vitro and in vivo.

[0051] However, activity of these MAbs was usually assessed againstnewly implanted rather than established tumors and was typicallysuperior to matching, but not optimal, doses of the unconjugated drug.

[0052] Although conjugates have been described with anti-tumor activityagainst established tumors that were superior to that of an optimal doseof unconjugated drug, the therapeutic index was low and superioractivity was achieved only at or near the maximum tolerated dose (MTD)of the conjugate (P. A. Trail et al., Cancer Res. 52, 5693 (1992)).

[0053] The results of clinical studies of drug and toxin conjugates(i.e., immunoconjugates) have also been disappointing, particularly forsolid tumors (E. S. Vitetta, R. J. Fulton, R. D. May, M. Till, J. W.Uhr, Science 238, 1098 (1987); H. G. Eichler, Biotherapy 3, 11 (1991);E. Wawrzynczak, Br. J Cancer 64, 624 (1991); G. A. Pietersz and I. F. C.McKenzie, Immunol. Rev. 129, 57 (1992)).

[0054] Very few antibodies are able to kill tumor cells by themselves,that is, in the absence of effector cells or complement as inantibody-dependent cellular cytotoxicity or complement-dependentcytotoxicity. BR96 is such an antibody, because it can kill cells byitself at an antibody concentration of approximately 10 μg/ml or higher.Such antibodies are of particular interest since they can interfere withsome key event in the survival of neoplastic cells.

[0055] Presently, chemotherapeutic agents, by themselves, do notdistinguish between malignant and normal cells. They are absorbed byboth cell types. Tumors that are detected early on such as acutelymphocytic leukemia and lymphomas are highly susceptible to drugs.

[0056] Tumors that are hidden until growth has reached a plateau, suchas cancer of the lung and colon, have little sensitivity to drugs.Normal cells with high growth fraction are inevitably attacked bytoday's anti-cancer drugs, explaining the prevalence of severe sideeffects in the gastrointestinal tract and of hair loss. This holds truewhether the cytotoxicity of the drug is due to alkylation,intercalation, or disruption of biosynthesis/antimetabolites.

[0057] The molecules of the invention, e.g., the immunotoxins, arehomogeneous molecules that retain the specificity of the cell bindingportion with the cytotoxic potential of the toxin.

[0058] It is thus apparent that antibodies, antibody conjugates andimmunotoxins that display a high degree of selectivity to a wide rangeof carcinomas, have anti-tumor activity, and are capable of beingreadily internalized by tumor cells, may be of great benefit in tumortherapy.

SUMMARY OF THE INVENTION

[0059] The present invention provides internalizing antibodies, antibodyconjugates and recombinant, single-chain-immunotoxins that are highlyselective for a range of human carcinomas. More specifically, the novelantibodies of the invention, designated as BR96 antibodies, are a murinemonoclonal antibody and a chimeric antibody that bind to a cell membraneantigen found on human carcinoma cells.

[0060] The novel conjugates and single-chain immunotoxins contain anexotoxin such as PE and bind to the antigen on tumor cells. Theantibodies, conjugates and single-chain immunotoxins are highly reactivewith carcinoma cells, such as those derived from breast, lung, colon andovarian carcinomas, showing no or limited reactivity with normal humancells or other types of tumors such as lymphomas or sarcomas. Inaddition, the antibodies of the invention internalize within thecarcinoma cells to which they bind and they are capable of killing tumorcells by themselves, i.e., not in conjugated form, and without effectorcells or complement.

[0061] Thus the BR96 antibodies are of particular use in therapeuticapplications, for example to react with tumor cells, and in conjugatesand single-chain immunotoxins as target-selective carriers of variousagents which have antitumor effects including chemotherapeutic drugs,toxins, immunological response modifiers, enzymes and radioisotopes. Theantibodies can thus be used as a component of various immunoconjugatesincluding antibody-drug and antibody-toxin conjugates, including ricinand PE-antibodies and ricin and PE-antibody fragment immunotoxins, whereinternalization of the conjugate is favored, and after radiolabeling todeliver radioisotope to tumors. The BR96 antibodies can also betherapeutically beneficial even in the unmodified form. Furthermore, theantibodies are useful for in vitro or in vivo diagnostic methodsdesigned to detect carcinomas.

BRIEF DESCRIPTION OF THE FIGURES

[0062]FIG. 1 depicts the percent inhibition of thymidine incorporationinto the DNA of 3396 breast carcinoma cells treated with a BR96-RAimmunotoxin at varying concentrations as described in Example 3, infra.BR6-RA is an internalizing antibody which is used as a negative controlbecause it does not bind to the 3396 cells.

[0063]FIG. 2 depicts the percent inhibition of thymidine incorporationinto the DNA of 2707 lung carcinoma cells treated with a BR96-RAimmunotoxin at varying concentrations as described in Example 3, infra.BR6-RA is an internalizing antibody which also binds to the 2707 cells.

[0064]FIG. 3 depicts the percent inhibition of thymidine incorporationinto the DNA of HCT116 colon carcinoma cells treated with a BR96-RAimmunotoxin at varying concentrations as described in Example 3, infra.BR96 does not bind to HCT 116 cells.

[0065]FIG. 4 depicts the percent inhibition of thymidine incorporationinto the DNA of C colon carcinoma cells treated with a BR96-RAimmunotoxin at varying concentrations as described in Example 3, infra.BR6-RA does not bind to the C cells; L6-RA binds to the C cells but doesnot internalize.

[0066]FIG. 5 depicts the percent inhibition of thymidine incorporationinto the DNA of 3347 colon carcinoma cells treated with a BR96-RAimmunotoxin at varying concentrations as described in Example 3, infra.BR96 does not bind to these cells while BR6 does.

[0067]FIG. 6 depicts the results of FACS analysis of the cytotoxicity ofpropidium iodide stained 3396 breast carcinoma cells, 2987 lungcarcinoma cells and 3619 colon carcinoma cells, as described in Example4, infra.

[0068]FIG. 7 depicts the effects of BR96 on cell proliferation ofvarious cell lines as described in Example 4, infra.

[0069]FIG. 8 illustrates the effect of BR96 on cell growth of variouscell lines, measured by a staining method as described in Example 4,infra.

[0070]FIG. 9 illustrates the results of tests to determineantibody-dependent cellular cytotoxicity activity of BR96 as describedin Example 5, infra.

[0071]FIG. 10 describes the results of tests to determinecomplement-dependent cytotoxicity activity of BR 96 as described inExample 6, infra.

[0072]FIG. 11 is a bar graph of the results of testing the reactivity ofBR96 against glycolipids as described in Example 7, infra.

[0073]FIG. 12 is a bar graph of the results of testing the reactivity ofBR96 against neoglycoproteins as described in Example 7, infra.

[0074]FIG. 13 is a graph of the binding activity of BR96 F(ab′)₂fragments compared to that of whole BR96 monoclonal antibody in an ELISAusing goat anti-K light chain detecting reagent, as described in Example8, infra.

[0075]FIG. 14 is a graph of the binding activity of BR96 F(ab′)₂fragments as compared to that of whole BR96 monoclonal antibody in anELISA using peroxidase conjugated protein A detecting reagent, asdescribed in Example 8, infra.

[0076]FIG. 15 is a diagram of vector phγ₁HC-D used in theelectroporation procedure, as described in Example 9, infra.

[0077]FIG. 16 is a diagram of vector PSV₂gpt/C_(K) used in theelectroporation procedure, as described in Example 9, infra.

[0078]FIG. 17 is a graph depicting the results of the competitionbinding assay comparing the binding of the murine BR96 monoclonalantibody of the invention with binding of the chimeric BR96 antibody ofthe invention, as described in Example 9, infra.

[0079]FIG. 18 depicts the results of FACS analysis of the cytotoxicityof the antibodies of the invention on 3396 breast carcinoma cells asdescribed in Example 10, infra.

[0080]FIG. 19 depicts the results of FACS analysis of the cytotoxicityof the antibodies of the invention on 2987 human lung adenocarcinomacells as described in Example 10, infra.

[0081]FIG. 20 depicts the results of FACS analysis of the cytotoxicityof the antibodies of the invention on MCF-7 cells as described inExample 10, infra.

[0082]FIG. 21 depicts the percent inhibition of thymidine incorporationinto the DNA of 3396 breast carcinoma cells treated with a murineBR96-RA immunotoxin and chimeric (Chi)BR96-RA at varying concentrationsas described in Example 10, infra.

[0083]FIG. 22 depicts the percent inhibition of thymidine incorporationinto the DNA of 3630 breast carcinoma cells treated with a murineBR96-RA immunotoxin and ChiBR96-RA at varying concentrations, asdescribed in Example 10, infra.

[0084]FIG. 23 is a graph depicting the antitumor effects of unmodifiedBR96 on the tumor cell line H2987, as described in Example 11, infra.

[0085]FIG. 24 is a bar graph illustrating the absence of tumors at theend of treatment for animals treated with BR96, as described in Example11, infra.

[0086]FIG. 25 depicts the dose effects of BR96 antibody afterimplantation of H2707 cells, as determined by tumor volume, as describedin Example 11, infra.

[0087]FIG. 26 illustrates the effects of treatment with F(ab′)₂fragments and chimeric BR96 after implantation of 2707 cells asdetermined by tumor volume, as described in Example 11, infra.

[0088]FIG. 27 illustrates the absence of tumors after treatment withvarious doses of BR96 antibody, as compared to the effects of F(ab′)₂fragments and chimeric BR96, as described in Example 11, infra.

[0089]FIG. 28 is a photograph of the gel obtained from non-reducingSDS-PAGE analysis of conjugated and unconjugated ChiBR96 IgG, Fab′ andF(ab′)₂ immunotoxins as described in Example 13, infra (Lane 1: ChiBR96IgG; Lane 2: ChiBR96 Fab′; Lane 3: ChiBR96 Fab′-LysPE40; Lane 4: NativePE; Lane 5: LysPE40; Lane 6: ChiBR96 IgG-LysPE40; Lane 7:ChiBR96(Fab′)₂; Lane 8: ChiBR96 F(ab′)₂-LysPE40; Lane 9: ChiBR96IgG-LysPE40; Lane 10: ChiBR96 IgG).

[0090]FIG. 29 is a graph depicting the results of competition ofChiBR96-PE and ChiBR96-LysPE40 binding as described in Example 13, infra(ChiBR96 (closed circle); ChiBR96-PE (closed square); ChiBR96-LysPE40(open triangle)).

[0091]FIGS. 30A, B, C are graphs of the direct binding of intactChiBR96-LysPE40, F(ab′)₂-LysPE40 and Fab′-LysPE40 to L2987 cells, asdescribed in Example 13, infra (ChiBR96 (closed circle); ChiBR96-LysPE40(open triangle); ChiBR96 F(ab′)₂ (open square); ChiBR96 F(ab′)₂ LysPE40(closed circle); ChiBR96 Fab′ (open circle); ChiBR96 Fab′-LysPE40 (opencircle)).

[0092]FIGS. 31A and B are graphs showing the determination ofendocytosis of cell-surface immunotoxin after modulation with ChiBR96-PEor ChiBR96-LysPE40 immunotoxins as described in Example 13, infra (31A:loss of cell surface immunotoxin under modulating and non-modulatingconditions; 31B: internalization of cell-bound immunotoxin usingimmunotoxin plus radiolabeled M-40/1 complex; ChiBR96-PE coated cellswere incubated at 4° C. (open circle) or 37° C. (closed circle);ChiBR96-LysPE40 coated cells were incubated at 4° C. (open triangle) or37° C. (closed triangle).

[0093]FIG. 32 is a graph of the cytotoxic effects of various ChiBR96forms conjugated to LysPE40 against MCF-7 cells as described in Example13, infra (ChiBR96-PE40 (closed square); ChiBR96 F(ab′)₂-PE40 (closedcircle); ChiBR96 Fab′-PE40 (closed triangle); PE40 open circle).

[0094]FIG. 33 is a bar graph depicting the results of competitionanalysis of ChiBR96-PE40 cytotoxic activity against MCF-7 cells asdescribed in Example 13, infra.

[0095]FIGS. 34A and B are graphs showing the results of proteinsynthesis inhibition analysis of ChiBR96-(PE/LysPE40) vs. PE againstMCF-7 cells as described in Example 13, infra (34A: 1 hr; 34B: 20hr;ChiBR96-PE (closed square); BR96-PE40 (closed circle); PE (closedtriangle).

[0096]FIG. 35 (SEQ ID NO: 3) is the DNA and amino acid sequence for BR96sFv encoded by plasmid pBR96 Fv, as described in Example 14, infra.

[0097]FIG. 36 is a schematic illustration of the construction ofexpression plasmid pBW 7.0 encoding BR96 sFv-PE40 as described inExample 14, infra (E, Eco RI; H, Hind III; K, KPNI; N, NDe I; S, Sal I;(Gly₄Ser)₃ represents a 15 amino acid linker).

[0098]FIG. 37A, B, C illustrate the purification of BR96 sFv-PE40 by gelfiltration as described in Example 14, infra (FIG. 37A: profile of gelfiltration column chromatography of renatured BR96 sFv-PE40 afterinitial purification over Q-Sepharose; FIG. 37B: 12% denaturingSDS-polyacrylamide gel stained with Coomassie brilliant blue; FIG. 37C:immunoblot of a 4-12% non-denaturing SDS-polyacrylamide gel probed withBR96 anti-idiotypic antibody; lanes 1-15 correspond to fractions 7-21 onthe gel filtration profile shown in FIG. 37A; lane M representsmolecular weight marker proteins in kilodaltons. Molecular weightstandards correspond to 670 kDa, 158 kDa, 44 kDa and 17 kDa eluted infractions 10, 15, 21 and 30, respectively).

[0099]FIG. 38 is a graph depicting the results of a direct binding assayon ELISA plates coated with Lewis-Y antigen and probed with BR96anti-idiotype antibody, and comparing the binding of BR96 IgG (opensquare), BR96 sFv-PE40 monomers (closed circle), BR96 sFv-PE40aggregates (closed triangle) and L6 IgG (open circle), as described inExample 14, infra.

[0100]FIG. 39 is a graph showing the results of binding analysis of BR96sFv-PE40, with competition of ¹²⁵ I-labeled BR96 IgG with BR96 sFv-PE40(closed circle), BR96 IgG (open square) and L6 IgG (open circle), asdescribed in Example 14, infra.

[0101]FIG. 40 is a graph showing the results of cytotoxicity analysis ofBR96 sFv-PE40 inhibition of protein synthesis in MCF-7 cells asdescribed in Example 14, infra (BR96 sFv-PE40 (closed circle) andChiBR96-LysPE40 (closed square)).

[0102]FIG. 41 are histograms of FACS analysis of five human carcinomalines as described in Example 14, infra (data is displayed in eachhistogram as the mean channel number for BR96 IgG or a human IgG controlantibody. Fluorescence intensity for each cell line is determined bysubtracting the human IgG mean channel number from the BR96 mean channelnumber).

[0103]FIG. 42 is a bar graph showing the results of competitivecytotoxic analysis of BR96 sFv-PE40 inhibition of protein synthesis inL2987 cells by BR96 sFv-PE40 (50 ng/ml) alone or in the presence ofeither BR96 IgG or L6 IgG (100 μg/ml) as described in Example 14, infra.

[0104]FIG. 43 is a graph showing anti-tumor effects of BR96-sFvPE40 invivo against MCF-7 human breast tumor xenografts, as described inExample 15, infra (ADM 6 mg/kg (closed square), BR96-sFvPE40 0.50 mg/kg(closed circle), BR96-sFvPE40 0.75 mg/kg (open circle), control (closedtriangle)).

[0105]FIG. 44 is a graph showing anti-tumor activity ofBR96-immunotoxins against L2987 human lung tumor xenografts as describedin Example 15, infra (sFv-PE40 0.125 mg/kg (closed triangle), sFv-PE400.25 mg/kg (closed circle), sFv-PE40 0.375 mg/kg (open circle), IgGLys-PE40 conjugate 1.25 mg/kg (closed square), Interleukin 6-PE40 0.375mg/kg (open square), control (closed triangle)).

[0106]FIG. 45 is a drawing of the structure of BR96-DOX.

[0107] FIGS. 46A-D are line graphs showing the antigen-specificantitumor activity of BR96-DOX.

[0108] (A) Control animals (closed square); animals treated withBR96-DOX (5 mg/kg DOX) (closed circle), IgG-DOX (5 mg/kg DOX) (closedtriangle); or optimized DOX (8 mg/kg) (open square).

[0109] (B) Control animals (closed square); animals treated with2BR96-DOX (10 mg/kg) (closed circle), IgG-DOX (10 mg/kg) (closedtriangle), or optimized DOX (8 mg/kg) (open square).

[0110] (C) Control animals (closed square); animals treated withBR96-DOX (5 mg/kg) (closed circle), IgG-DOX (5 mg/kg) (closed triangle),or DOX (6 mg/kg) (open square).

[0111] (D) Control animals (closed square) animals treated with BR96-DOX(8 mg/kg) (closed circle), or DOX (8 mg/kg) (open triangle).

[0112]FIG. 47 is a line graph showing that BR96-DOX cures athymic miceof large disseminated tumors. Untreated controls (closed triangle),BR96-DOX treated (8 mg/kg) (closed circle) or DOX treated (8 mg/kg)(closed square) 82, 86 and 90 days after inoculation of tumor cells.

[0113]FIG. 48 is a line graph showing that BR96-DOX cures human lungtumors implanted in athymic rats. Control animals (closed square),animals treated with BR96-DOX (4 mg/kg) (open circle), BR96-DOX (2mg/kg) (closed triangle), or DOX (4 mg/kg) (open square).

[0114]FIG. 49(a) provides a synthetic scheme for preparing a thiolatedantibody using SPDP as the thiolation agent.

[0115]FIG. 49(b) provides a synthetic scheme for preparing animmunoconjugate of the invention in which the ligand is a SPDP-thiolatedantibody.

[0116]FIG. 49(c) provides a synthetic scheme for preparing animmunoconjugate of the invention in which the ligand is animinothiolane-thiolated antibody.

[0117]FIG. 50 shows a process for reducing with DTT an antibody toprepare a “relaxed” antibody and synthesis of an immunoconjugate of theinvention.

[0118]FIG. 51 provides in vitro cytotoxic activity data forBR64-Adriamycin conjugates of the invention against L2987 tumors.

[0119]FIG. 52 provides in vivo cytotoxic activity data forBR64-Adriamycin conjugates of the invention against L2987 tumors.

[0120] FIGS. 53A/B/C provides comparative in vivo cytotoxic data forcombination therapy using BR64, Adriamycin and non-binding conjugate(SN7-Adriamycin).

[0121]FIG. 54 provides in vivo cytotoxic activity data forBombesin-Adriamycin conjugates of the invention against H345 tumors.

[0122]FIG. 55 provides in vitro cytotoxic activity data for Adriamycinconjugates of relaxed chimeric BR96 and SPDP-thiolated chimeric BR96.

[0123]FIG. 56 provides in vivo cytotoxic activity data for Adriamycinconjugates of relaxed BR64 and relaxed chimeric L6 against L2987 tumors.

[0124] FIGS. 57 to 59 provide in vivo cytotoxic activity data againstL2987 tumors for Adriamycin conjugates of relaxed chimeric BR96 comparedto free Adriamycin and non-binding conjugates.

[0125]FIG. 60 provides in vivo cytotoxic activity data for Adriamycinconjugates of relaxed chimeric BR96 against RCA Human Breast Tumors.

[0126]FIG. 61 provides in vivo cytotoxic activity data for Adriamycinconjugates of relaxed chimeric BR96 against RCA Human Colon Tumors.

[0127]FIG. 62 provides a graph of the effect on —SH titer as a functionof mole ratio of DTT to antibody in the preparation, under an inertatmosphere, of a relaxed antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0128] Definitions

[0129] As used in this application, the following words or phrases havethe meanings specified.

[0130] As used herein, “functional equivalent” means being capable of(1) binding the antigen binding site to which BR96 is directed (i.e.,competitively inhibit the antigen binding site), (2) binding carcinomacells, (3) being internalized within the carcinoma cells to which theybind, and/or (4) mediating ADCC and CDC effector functions.

[0131] As used herein, “joined” means to couple directly or indirectlyone molecule with another by whatever means, e.g., by covalent bonding,by non-covalent bonding, by ionic bonding, or by non-ionic bonding.Covalent bonding includes bonding by various linkers such as thioetherlinkers or thioester linkers. Direct coupling involves one moleculeattached to the molecule of interest. Indirect coupling involves onemolecule attached to another molecule not of interest which in turn isattached directly or indirectly to the molecule of interest.

[0132] As used herein, “recombinant molecule” means a molecule producedby genetic engineering methods.

[0133] As used herein, “fragment” is defined as at least a portion ofthe variable region of the immunoglobulin molecule which binds to itstarget, i.e., the antigen binding region. Some of the constant region ofthe immunoglobulin may be included.

[0134] As used herein, an “immunoconjugate” means any molecule or ligandsuch as an antibody or growth factor chemically or biologically linkedto a cytotoxin, a radioactive agent, an anti-tumor drug or a therapeuticagent. The antibody or growth factor may be linked to the cytotoxin,radioactive agent, anti-tumor drug or therapeutic agent at any locationalong the molecule so long as it is able to bind its target. Examples ofimmunoconjugates include immunotoxins and antibody conjugates.

[0135] As used herein, “selectively killing” means killing those cellsto which the antibody binds.

[0136] As used herein, examples of “carcinomas” include bladder, breast,colon, liver, lung, ovarian, and pancreatic carcinomas.

[0137] As used herein, “immunotoxin” means an antibody or growth factorchemically or biologically linked to a cytotoxin or cytotoxic agent.

[0138] As used herein, an “effective amount” is an amount of theantibody, immunoconjugate, recombinant molecule which kills cells orinhibits the proliferation thereof.

[0139] As used herein, “competitively inhibits” means being capable ofbinding to the same target as another molecule. With regard to anantibody, competitively inhibits mean that the antibody is capable ofrecognizing and binding the same antigen binding region to which anotherantibody is directed.

[0140] As used herein, “antigen-binding region” means that part of theantibody, recombinant molecule, the fusion protein, or theimmunoconjugate of the invention which recognizes the target or portionsthereof.

[0141] As used herein, “therapeutic agent” means any agent useful fortherapy including anti-tumor drugs, cytotoxins, cytotoxin agents, andradioactive agents.

[0142] As used herein, “anti-tumor drug” means any agent useful tocombat cancer including, but not limited to, cytotoxins and agents suchas antimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, mytotane (0,P′-(DDD)), interferons and radioactiveagents.

[0143] As used herein, “a cytotoxin or cytotoxic agent” means any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

[0144] As used herein, “a radioactive agent” includes any radioisotopewhich is effective in destroying a tumor. Examples include, but are notlimited to, cobalt-60 and X-rays. Additionally, naturally occurringradioactive elements such as uranium, radium, and thorium whichtypically represent mixtures of radioisotopes, are suitable examples ofa radioactive agent.

[0145] As used herein, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular or subcutaneous administration, or theimplantation of a slow-release device such as a miniosmotic pump, to thesubject.

[0146] As used herein, “directly” means the use of antibodies coupled toa label. The specimen is incubated with the labeled antibody, unboundantibody is removed by washing, and the specimen may be examined.

[0147] As used herein, “indirectly” means incubating the specimen withan unconjugated antibody, washing and incubating with afluorochrome-conjugated antibody. The second or “sandwich” antibody thusreveals the presence of the first.

[0148] As used herein “reacting” means to recognize and bind the target.The binding may be non-specific. Specific binding is preferred.

[0149] As used herein, “curing” means to provide substantially completetumor regression so that the tumor is not palpable for a period of time,i.e., ≧10 tumor volume doubling delays (TVDD=the time in days that ittakes for control tumors to double in size).

[0150] As used herein, “tumor associated antigens” means any cellsurface antigen which is generally associated with tumor cells, i.e.,occurring to a greater extent as compared with normal cells. Suchantigens may be tumor specific. Alternatively, such antigens may befound on the cell surface of both tumorigenic and non-tumorigenic cells.These antigens need not be tumor specific. However, they are generallymore frequently associated with tumor cells than they are associatedwith normal cells.

[0151] As used herein, “tumor targeted antibody” means any antibodywhich recognizes cell surface antigens on tumor (i.e., cancer) cells.Although such antibodies need not be tumor specific, they are tumorselective, i.e., bind tumor cells more so than it does normal cells.

[0152] As used herein, “internalizing tumor targeted antibody” includesany tumor targeted antibody which is easily taken up by the tumor cellsto which they bind.

[0153] As used herein, “internalizing tumor targeted antibody whichrecognizes the Le^(y) determinant” includes internalizing tumor targetedantibody which specifically recognizes at least a portion of the Le^(y)determinant.

[0154] As used herein, “inhibit proliferation” means to interfere withcell growth by whatever means.

[0155] As used herein, “mammalian tumor cells” include cells fromanimals such as human, ovine, porcine, murine, bovine animals.

[0156] As used herein, “pharmaceutically acceptable carrier” includesany material which when combined with the antibody retains theantibody's immunogenicity and non-reactive with the subject's immunesystems. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Other carriers may also include sterile solutions,tablets including coated tablets and capsules.

[0157] Typically such carriers contain excipients such as starch, milk,sugar, certain types of clay, gelatin, stearic acid or salts thereof,magnesium or calcium stearate, talc, vegetable fats or oils, gums,glycols, or other known excipients. Such carriers may also includeflavor and color additives or other ingredients. Compositions comprisingsuch carriers are formulated by well known conventional methods.

[0158] In order that the invention herein described may be more fullyunderstood, the following description is set forth.

[0159] 1. Novel Antibodies of the Invention

[0160] The present invention relates to novel antibodies that are highlyspecific for carcinoma cells. More particularly, the antibodies reactwith a range of carcinomas such as breast, lung, ovary and coloncarcinomas, while showing none or limited reactivity with normal humantissues or other types of tumors such as sarcomas or lymphomas.

[0161] One type of novel antibodies of the invention is designated BR96.The BR96 antibodies can be used to isolate and characterize the antigento which they bind. Thus, the BR96 antibodies can be used as a probe toidentify and characterize the epitope recognized and to further definethe cell membrane antigen with which they react (see, e.g., Nudelman etal., “Characterization of Human Melanoma-Associated Ganglioside AntigenDefined By A Monoclonal Antibody, 4.2,” J. Biol. Chem., 257(1):12752-56(1982) and Hakomori, “Tumor Associated Carbohydrate Antigens,” Ann. Rev.Immunol. 2:103-262(1984)).

[0162] BR96 recognizes as at least part of its binding site a portion ofan epitope of a Le^(y) carbohydrate determinant which is a portion of anantigen abundantly expressed on carcinomas of the colon, breast, ovary,and lung and, to a lesser extent, on epithelial cells from thegastrointestinal tract. Further, BR96 in the absence of effector cellsor complement can inhibit tumor cell DNA synthesis.

[0163] Results of preliminary epitope screens conducted on monoclonalantibody BR96 have indicated that the epitope which is a portion of theantigen on the carcinoma cells to which BR96 antibody binds is afucosylated variant of Lewis Y (Le^(y)). Le^(y) has been described byAbe et al., J. Biol. Chem. 258:8934 (1983); Lloyd et al., Immunogenetics17:537 (1983); Brown et al., Biosci. Rep. 3:163 (1983); and Hellstrom etal., Cancer Res. 46:3917 (1986). Certain fucosylated variants of Lewis Yhave been described by Abe et al., Cancer Res. 46:2639-2644 (1986).

[0164] The monoclonal antibody of the invention can be produced usingwell-established hybridoma techniques first introduced by Kohler andMilstein (see, Kohler and Milstein, “Continuous Cultures Of Fused CellsSecreting Antibody Of Pre-Defined Specificity,” Nature, 256:495-97(1975). See, also, Brown et al., “Structural Characterization Of HumanMelanoma-Associated Antigen p97 with Monoclonal Antibodies,” J. Immunol.127(2):539-46 (1981)); Brown et al., “Protein Antigens Of Normal AndMalignant Human Cells Identified By Immunoprecipitation With MonoclonalAntibodies,” J. Biol. Chem. 255:4980-83 (1980); Yeh et al., “CellSurface Antigens Of Human Melanoma Identified By Monoclonal Antibody,”Proc. Natl. Acad. Sci. USA, 76(6):297-31 (1979); and Yeh et al., “ACell-Surface Antigen Which is Present In the Ganglioside Fraction AndShared By Human Melanomas,” Int. J. Cancer. 29:269-75 (1982).

[0165] These techniques involve the injection of an immunogen (e.g.,cells or cellular extracts carrying the antigen or purified antigen)into an animal (e.g., a mouse) so as to elicit a desired immune response(i.e., antibodies) in that animal. After a sufficient time,antibody-producing lymphocytes are obtained from the animal either fromthe spleen, lymph nodes or peripheral blood. Preferably, the lymphocytesare obtained from the spleen. The splenic lymphocytes are then fusedwith a myeloma cell line, usually in the presence of a fusing agent suchas polyethylene glycol (PEG). Any of a number of myeloma cell lines maybe used as a fusion partner according to standard techniques; forexample, the P3-NS1/1Ag4-1, P3-x63-Ag8.653 or Sp2/O Ag14 myeloma lines.These myeloma lines are available from the American Type CultureCollection (“ATCC”) in Rockville, Md.

[0166] The resulting cells, which include the desired hybridomas, arethen grown in a selective medium, such as HAT medium, in which unfusedparental myeloma or lymphocyte cells eventually die. Only the hybridomacells survive and can be grown under limiting conditions to obtainisolated clones. The supernatants of the hybridomas are screened for thepresence of antibody of that desired specificity, e.g., by immunoassaytechniques using the antigen that had been used for immunization.Positive clones can then be subcloned under limiting dilution conditionsand the monoclonal antibody produced can be isolated. Hybridomasproduced according to these methods can be propagated in vitro or invivo (in ascites fluid) using techniques known in the art (see,generally, Fink et al., supra at page 123, FIGS. 6-11). Commonly usedmethods for purifying monoclonal antibodies include ammonium sulfateprecipitation, ion exchange chromatography, and affinity chromatography(see, e.g., Zola et al., “Techniques For The Production AndCharacterization Of Monoclonal Hybridoma Antibodies,” in MonoclonalHybridoma Antibodies: Techniques And Applications, Hurell (ed.), pp.51-52 (CRC Press 1982)).

[0167] According to a preferred embodiment, a monoclonal antibody ofthis invention, designated BR96, was produced via the hybridomatechniques described hereinbelow using a breast cancer cell line 3396 asthe immunogen. The BR96 hybridoma, prepared as described hereinbelow andproducing the BR96 antibody, was deposited on Feb. 22, 1989 with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 and has there been identified as follows:

[0168] BR96 ATCC Accession No.: HB 10036

[0169] The BR96 antibody is of the IgG3 subclass. The antibody displaysa high specificity for carcinoma cells of different organ types, forexample, tumors of the breast, lung, colon and ovary as well as culturedcell lines established from various breast, lung and colon carcinomas.Furthermore, the BR96 antibody shows no binding to other types of tumorcells such as the T-cell lymphoma cells lines, CEM and MOLT-4, the Bcell lymphoma cell line P3HR-1 or melanoma cell lines. The BR96 antibodyis able to be internalized in antigen-positive tumor cells, is toxic toantigen-positive tumor cells, mediates antibody-dependent cellularcytotoxicity and complement-dependent cytotoxicity activity, andsurprisingly, is cytotoxic alone, i.e., in unmodified form. The BR96antibodies appear to recognize a novel epitope of the Le^(y)determinant.

[0170] The present invention provides an immunoconjugate comprising amolecule having the antigen-binding region of the BR96 monoclonalantibody joined to doxorubicin. It would be clear that doxorubicin maybe joined at any location along the molecule so long as it retains itsability to bind its target. Doxorubicin may be joined by any meansincluding chemical and biological means.

[0171] Clearly analogs and homologs of doxorubicin are encompassed bythe invention. For example, an improved analog of doxorubicin isFe-chelate.

[0172] 2. Fragments of the Monoclonal Antibodies of the Invention

[0173] According to another embodiment, F(ab′)₂ fragments of the BR96monoclonal antibody were produced by pepsin digestion of purified BR96(Lamoyi, “Preparation of F(ab′)₂ Fragments from Mouse IgG of VariousSubclasses,” Methods of Enzymol. 121:652-663 (1986)), as describedhereinbelow. The binding of the F(ab′)₂ fragments to tumor (3396) andMCF7 cells was shown to be comparable to the binding of the whole BR96monoclonal antibody.

[0174] 3. Chimeric Antibodies of the Invention

[0175] In another preferred embodiment, a chimeric (murine/human)antibody of the invention was produced using a two-step homologousrecombination procedure as described by Fell et al., in Proc. Natl.Acad. Sci. USA 86:8507-8511 (1989) and in co-pending patent applicationU.S. Ser. No. 243,873, filed Sep. 14, 1988, and Ser. No. 468,035, filedJun. 22, 1990, assigned to the same assignee as the present application;the disclosures of all of these documents are incorporated in theirentirety by reference herein. This two-step protocol involves use of atarget vector encoding human IgGγ1 heavy chain to transfect a mousehybridoma cell line expressing murine BR96 monoclonal antibody(hybridoma ATCC No. HB 10036) to produce a hybridoma expressing a BR96chimeric antibody containing human IgGγ1 heavy chain. This hybridoma isthen transfected with a target vector containing DNA encoding humankappa (K) light chain to produce a murine hybridoma expressing a BR96chimeric antibody containing human IgGγ1 heavy chain and human K lightchain. The target vectors used to transfect the hybridomas are thephγ1HC-D vector digested with Xbal enzyme (Bristol-Myers Squibb Co.,Seattle, Wash., NRRL No. B 18599) and the HindIII digested pSV₂gpt/C_(K)vector (Bristol-Myers Squibb Co., Seattle, Wash., NRRL No. B 18507).

[0176] The chimeric BR96 hybridoma, identified herein as ChiBR96,prepared as described hereinbelow and producing the chimerichuman/murine BR96 antibody, was deposited on May 23, 1990, with theATCC, 12301 Parklawn Drive, Rockville, Md. 20852 and has there beenidentified as follows.

[0177] ChiBR96 ATCC Accession No.: HB 10460

[0178] Once the hybridoma that expresses the chimeric antibody isidentified, the hybridoma is cultured and the desired chimeric moleculesare isolated from the cell culture supernatant using techniques wellknown in the art for isolating monoclonal antibodies.

[0179] The term “BR96 antibody” as used herein includes whole, intactpolyclonal and monoclonal antibody materials such as the murine BR96monoclonal antibody produced by hybridoma ATCC No. HB 10036, andchimeric antibody molecules such as chimeric BR96 antibody produced byhybridoma ATCC No. 10460. The BR96 antibody described above includes anyfragments thereof containing the active antigen-binding region of theantibody such as Fab, F(ab′)₂ and Fv fragments, using techniques wellestablished in the art (see, e.g., Rousseaux et al., “Optimal ConditionsFor The Preparation of Proteolytic Fragments From Monoclonal IgG ofDifferent Rat IgG Subclasses,” in Methods Enzymol., 121:663-69 (AcademicPress 1986)). The BR96 antibody of the invention also includes fusionproteins.

[0180] In addition, the BR96 antibody of this invention does not displayany immunohistologically detectable binding to normal human tissues frommajor organs, such as kidney, spleen, liver, skin, lung, breast, colon,brain, thyroid, heart, lymph nodes or ovary. Nor does the antibody reactwith peripheral blood leukocytes. BR96 antibody displays limited bindingto some cells in the tonsils and testes, and binds to acinar cells inthe pancreas, and to epithelial cells in the stomach and esophagus.Thus, the BR96 antibody is superior to most known antitumor antibodiesin the high degree of specificity for tumor cells as compared to normalcells (see, e.g., Helltrom et al., “Immunological Approaches To TumorTherapy: Monoclonal Antibodies, Tumor Vaccines, And Anti-Idiotypes,” inCovalently Modified Antigens And Antibodies In Diagnosis And Therapy,Quash/Rodwell (eds.), pp. 1-39 (Marcel Dekker, Inc., 1989) and Bagshawe,“Tumour Markers—Where Do We Go From Here,” Br. J Cancer. 48:167-75(1983)).

[0181] Also included within the scope of the invention areanti-idiotypic antibodies to the BR96 antibody of the invention. Theseanti-idiotypic antibodies can be produced using the BR96 antibody and/orthe fragments thereof as immunogen and are useful for diagnosticpurposes in detecting humoral response to tumors and in therapeuticapplications, e.g., in a vaccine, to induce an anti-tumor response inpatients (see, e.g., Nepom et al., “Anti-Idiotypic Antibodies And TheInduction Of Specific Tumor Immunity,” in Cancer And Metastasis Reviews,6:487-501 (1987)).

[0182] In addition, the present invention encompasses antibodies thatare capable of binding to the same antigenic determinant as the BR96antibodies and competing with the antibodies for binding at that site.These include antibodies having the same antigenic specificity as theBR96 antibodies but differing in species origin, isotype, bindingaffinity or biological functions (e.g., cytotoxicity). For example,class, isotype and other variants of the antibodies of the inventionhaving the antigen-binding region of the BR96 antibody can beconstructed using recombinant class-switching and fusion techniquesknown in the art (see, e.g., Thammana et al., “Immunoglobulin HeavyChain Class Switch From IgM to IgG In A Hybridoma,” Eur. J Immunol.13:614 (1983); Spira et al., “The Identification Of Monoclonal ClassSwitch Variants By Subselection And ELISA Assay,” J. Immunol. Meth.74:307-15 (1984); Neuberger et al., “Recombinant Antibodies PossessingNovel Effector Functions,” Nature. 312:604-608 (1984); and Oi et al.,“Chimeric Antibodies,” Biotechniques, 4(3):214-21 (1986)). Thus, otherchimeric antibodies or other recombinant antibodies (e.g., fusionproteins wherein the antibody is combined with a second protein such asa lymphokine or a tumor inhibitory growth factor) having the samebinding specificity as the BR96 antibodies fall within the scope of thisinvention.

[0183] Genetic engineering techniques known in the art are used asdescribed herein to prepare recombinant immunotoxins produced by fusingantigen binding regions of antibody BR96 to a therapeutic or cytotoxicagent at the DNA level and producing the cytotoxic molecule as achimeric protein.

[0184] Examples of therapeutic agents include, but are not limited to,antimetabolites, alkylating agents, anthracyclines, antibiotics, andanti-mitotic agents.

[0185] Antimetabolites include methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine.

[0186] Alkylating agents include mechlorethamine, thiotepa chlorambucil,melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide,busulfan, dibromomannitol, streptozotocin, mitomycin C, andcis-dichlorodiamine platinum (II) (DDP) cisplatin.

[0187] Anthracyclines include daunorubicin (formerly daunomycin) anddoxorubicin (also referred to herein as adriamycin). Additional examplesinclude mitozantrone and bisantrene.

[0188] Antibiotics include dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC).

[0189] Antimytotic agents include vincristine and vinblastine (which arecommonly referred to as vinca alkaloids).

[0190] Other cytotoxic agents include procarbazine, hydroxyurea,asparaginase, corticosteroids, mytotane (O, P′-(DDD)), interferons.

[0191] Further examples of cytotoxic agents include, but are not limitedto, ricin, doxorubicin, taxol, cytochalasin B, gramicidin D, ethidiumbromide, etoposide, tenoposide, colchicin, dihydroxy anthracin dione,1-dehydrotestosterone, and glucocorticoid.

[0192] Clearly analogs and homologs of such therapeutic and cytotoxicagents are encompassed by the present invention. For example, thechemotherapeutic agent aminopterin has a correlative improved analognamely methotrexate.

[0193] Further, the improved analog of doxorubicin is an Fe-chelate.Also, the improved analog for 1-methylnitrosourea is lomustine. Further,the improved analog of vinblastine is vincristine. Also, the improvedanalog of mechlorethamine is cyclophosphamide.

[0194] 4. Immunotoxins of the Invention

[0195] Recombinant immunotoxins, particularly single-chain immunotoxins,have an advantage over drug/antibody conjugates in that they are morereadily produced than these conjugates, and generate a population ofhomogenous molecules, i.e., single peptides composed of the same aminoacid residues.

[0196] The techniques for cloning and expressing DNA sequences encodingthe amino acid sequences corresponding to the single-chain immunotoxinBR96 sFv-PE40, e.g., synthesis of oligonucleotides, PCR, transformingcells, constructing vectors, expression systems, and the like arewell-established in the art, and most practitioners are familiar withthe standard resource materials for specific conditions and procedures(see, e.g., Sambrook et al., eds., Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)).

[0197] Details of the construction of the single-chain recombinantimmunotoxin of the invention, BR96 sFv-PE40 are provided in Example 13,infra. Briefly, polymerase chain reaction (PCR) (Mullis et al., U.S.Pat. Nos. 4,683,195 and 4,683,202; Mullis and Faloona, Methods Enzymol.154:335-350 (1987)) is used to amplify a 550 bp BR96 sFv sequence (FIG.35) encoded by plasmid pBR96 Fv using the selected primers.

[0198] After PCR amplification and enzymatic digestion the 550 bpfragment is ligated using standard procedures into a 4220 bp fragmentfrom vector pMS8 (Covell et al., Cancer Res. 46:3969-3978 (1986))encoding the gene for PE40 to form intermediate vector pBW 7.01.

[0199] A fragment from pBR96 Fv is then subcloned into pBW 7.01 to formplasmid pBW 7.0 encoding the BR96 sFv-PE40 gene fusion. Correctligations for vector construction are confirmed by DNA sequence analysisusing known procedures (Sanger et al., Proc. Natl. Acad. Sci. USA74:5463 (1977) and Messing et al., Nucleic Acids Res. 9:309 (1981)).Colonies are then screened by restriction enzyme digestion for theappropriate plasmids.

[0200] The following include preferred embodiments of theimmunoconjugates of the invention. Other embodiments which are known inthe art are encompassed by the invention. Specific embodiments are setforth in the Examples which follow.

[0201] The invention is not limited to these specific immunoconjugates,but also includes other immunoconjugates incorporating antibodies and/orantibody fragments according to the present invention.

[0202] The conjugates comprise at least one drug molecule connected by alinker of the invention to a targeting ligand molecule that is reactivewith the desired target cell population. The ligand molecule can be animmunoreactive protein such as an antibody, or fragment thereof, anon-immunoreactive protein or peptide ligand such as bombesin or, abinding ligand recognizing a cell associated receptor such as a lectinor steroid molecule.

[0203] As previously noted, a conjugate of the invention is representedby general Formula (I):

[0204] in which

[0205] D is a drug molecule;

[0206] n is 1 to 10;

[0207] p is 1 to 6;

[0208] Y is O or NH₂ ⁺C17;

[0209] z is 0 or 1;

[0210] q is about 1 to about 10;

[0211] X is a ligand; and,

[0212] A is Michael Addition Adduct.

[0213] For a better understanding of the invention, the drugs andligands will be discussed individually. The intermediates used for thepreparation of the conjugates and the synthesis of the conjugates thenwill be explained.

[0214] The Drug

[0215] One skilled in the art understands that the present inventionrequires the drug and ligand to be linked by means of an acylhydrazonelinkage, through a Michael Addition Adduct and thioether-containinglinker. Neither the specific drug nor the specific ligand is to beconstrued as a limitation on the present invention. The linkers of thepresent invention may be used with any drug having any desiredtherapeutic, biological activity-modifying or prophylactic purpose,limited only in that the drug used in preparing the conjugate be able toform an hydrazone bond. Preferably, to prepare the hydrazone, the drugshould have a reactively available carbonyl group, such as, for example,a reactive aldehyde or ketone moiety (represented herein as “[D-(C═O)]”)which is capable of forming a hydrazone (i.e., a —C═N—NH— linkage). Thedrug hydrazone linkage is represented herein as “[D═N—NH—.” In addition,the reaction of that reactively available group with the linkerpreferably must not destroy the ultimate therapeutic activity of theconjugate, whether that activity is the result of the drug beingreleased at the desired site of action or whether the intact conjugate,itself, is responsible for such activity.

[0216] One skilled in the art understands that for those drugs whichlack a reactively available carbonyl group, a derivative containing sucha carbonyl group may be prepared using procedures known in the art. Ascan be appreciated, the conjugate prepared from such derivatized drugmust retain therapeutic activity when present at the active site,whether this is due to the intact conjugate, or otherwise.Alternatively, the derivatized drug or, for example, a prodrug, must bereleased in such a form that a therapeutically active form of the drugis present at the active site.

[0217] The present linker invention may be used in connection with drugsof substantially all therapeutic classes including, for example,antibacterials, antivirals, antifungals, anticancer drugs,antimycoplasmals, and the like. The drug conjugates so constructed areeffective for the usual purposes for which the corresponding drugs areeffective, and have superior efficacy because of the ability, inherentin the ligand, to transport the drug to the desired cell where it is ofparticular benefit.

[0218] Further, because the conjugates of the invention can be used formodifying a given biological response, the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1, (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

[0219] The preferred drugs for use in the present invention arecytotoxic drugs, particularly those which are used for cancer therapy.Such drugs include, in general, alkylating agents, anti-proliferativeagents, tubulin binding agents and the like. Preferred classes ofcytotoxic agents include, for example, the anthracycline family ofdrugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, and thepodophyllotoxins. Particularly useful members of those classes include,for example, adriamycin, carminomycin, daunorubicin, aminopterin,methotrexate, methopterin, dichloromethotrexate, mitomycin C,porfiromycin, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside,podophyllotoxin, or podophyllotoxin derivatives such as etoposide oretoposide phosphate, melphalan, vinblastine, vincristine, leurosidine,vindesine, leurosine and the like. As noted previously, one skilled inthe art may make chemical modifications to the desired compound in orderto make reactions of that compound more convenient for purposes ofpreparing conjugates of the invention.

[0220] A highly preferred group of cytotoxic agents for use as drugs inthe present invention include drugs of the following formulae:

[0221] The Methotrexate Group of Formula (2)

[0222] in which

[0223] R¹² is amino or hydroxy;

[0224] R⁷ is hydrogen or methyl;

[0225] R⁸ is hydrogen, fluoro, chloro, bromo or iodo;

[0226] R⁹ is hydroxy or a moiety which completes a salt of thecarboxylic acid;

[0227] The Mitomycin Group of Formula (3)

[0228] in which R¹⁰ is hydrogen or methyl;

[0229] The Bleomycin Group of Formula (4)

[0230] in which R¹¹ is hydroxy, amino, C₁-C₃ alkylamino, di(C₁-C₃alkyl)amino, C₄-C₆ polymethylene amino,

[0231] Melphalan of Formula (5)

[0232] 6-Mercaptopurine of Formula (6)

[0233] A Cytosine Arabinoside of Formula (7)

[0234] The Podophyllotoxins of Formula (8)

[0235] in which

[0236] R¹³ is hydrogen or methyl;

[0237] R¹⁴ is methyl or thienyl;

[0238] or a phosphate salt thereof;

[0239] The Vinca Alkaloid Group of Drugs of Formula (9)

[0240] in which R¹⁵ is H, CH₃ or CHO; when R¹⁷ and R¹⁸ are taken singly,R¹⁸ is H, and one of R¹⁶ and R¹⁷ is ethyl and the other is H or OH; whenR¹⁷ and R¹⁸ are taken together with the carbons to which they areattached, they form an oxirane ring in which case R¹⁶ is ethyl, R¹⁹ ishydrogen, (C₁-C₃ alkyl)-CO, or chlorosubstituted (C₁-C₃ alkyl)-CO;

[0241] Difluoronucleosides of Formula (10)

[0242] in which R²¹ is a base of one of the formulae:

[0243] in which

[0244] R²² is hydrogen, methyl, bromo, fluoro, chloro or iodo;

[0245] R²³ is —OH or —NH₂;

[0246] R²⁴ is hydrogen, bromo, chloro or iodo;

[0247] or,

[0248] The Anthracyclines Antibiotics of Formula (11)

[0249] wherein

[0250] R₁ is —CH₃, —CH₂OH, —CH₂OCO(CH₂)₃CH₃ or —CH₂OCOCH(OC₂H₅)₂

[0251] R₃ is —OCH₃, —OH or —H

[0252] R⁴ is —NH₂, —NHCOCF₃, 4-morpholinyl, 3-cyano-4-morpholinyl,1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine,cyanomethylamine, or 1 -cyano-2-methoxyethyl amine

[0253] R₅ is —OH, —OTHP, or —H; and,

[0254] R₆ is —OH or —H provided that R₆ is not —OH when R₅ is —OH or—OTHP.

[0255] The most highly preferred drugs are the anthracycline antibioticagents of Formula (11), described previously. One skilled in the artunderstands that this structural formula includes compounds which aredrugs, or are derivatives of drugs, which have acquired in the artdifferent generic or trivial names. Table 14, which follows, representsa number of anthracycline drugs and their generic or trivial names andwhich are especially preferred for use in the present invention.

TABLE 14 Com- pound R₁ R₃ R₄ R₅ R₆ Daunor- CH₃ OCH₃ NH₂ OH H ubicin^(a)Adria- CH₂OH OCH₃ NH₂ OH H mycin^(b) Detor- CH₂OCOCH(OC₂H₅)₂ OCH₃ NH₂ OHH ubicin Car- CH₃ OH NH₂ OH H mino- mycin Idaru- CH₃ H NH₂ OH H bicinEpiru- CH₂OH OCH₃ NH₂ H OH bicin Esoru- CH₂OH OCH₃ NH₂ H H bicin THPCH₂OH OCH₃ NH₂ OTHP H AD-32 CH₂OCO(CH₂)₃CH₃ OCH₃ NHCOCF₃ OH H

[0256] Of the compounds shown in Table 14, the most highly preferreddrug is adriamycin. Adriamycin (also referred to herein as “ADM”) isthat anthracycline of Formula (11) in which R₁ is —CH₂OH, R₃ is —OCH₃,R₄ is —NH₂, R₅ —OH, and R₆ is —H.

[0257] The Ligands

[0258] One skilled in the art understands that “ligand” includes withinits scope any molecule that specifically binds or reactively associatesor complexes with a receptor or other receptive moiety associated with agiven target cell population. This cell reactive molecule, to which thedrug reagent is linked via the linker in the conjugate, can be anymolecule that binds to, complexes with or reacts with the cellpopulation sought to be therapeutically or otherwise biologicallymodified and, which possesses a free reactive sulfhydryl (—SH) group orcan be modified to contain such a sulfhydryl group. The cell reactivemolecule acts to deliver the therapeutically active drug moiety to theparticular target cell population with which the ligand reacts. Suchmolecules include, but are not limited to, large molecular weightproteins (generally greater than 10,000 daltons) such as, for example,antibodies, smaller molecular weight proteins (generally, less than10,000 daltons), polypeptide or peptide ligands, and non-peptidylligands.

[0259] The non-immunoreactive protein, polypeptide, or peptide ligandswhich can be used to form the conjugates of this invention may include,but are not limited to, transferrin, epidermal growth factors (“EGF”),bombesin, gastrin, gastrin-releasing peptide, platelet-derived growthfactor, IL-2, IL-6, tumor growth factors (“TGF”), such as TGF-α andTGF-β, vaccinia growth factor (“VGF”), insulin and insulin-like growthfactors I and II. Non-peptidyl ligands may include, for example,steroids, carbohydrates and lectins.

[0260] The immunoreactive ligands comprise an antigen-recognizingimmunoglobulin (also referred to as “antibody”), or antigen-recognizingfragment thereof. Particularly preferred immunoglobulins are thoseimmunoglobulins which can recognize a tumor-associated antigen. As used,“immunoglobulin” may refer to any recognized class or subclass ofimmunoglobulins such as IgG, IgA, IgM, IgD, or IgE. Preferred are thoseimmunoglobulins which fall within the IgG class of immunoglobulins. Theimmunoglobulin can be derived from any species. Preferably, however, theimunoglobulin is of human, murine, or rabbit origin. Further, theimmunoglobulin may be polyclonal or monoclonal, preferably monoclonal.

[0261] As noted, one skilled in the art will appreciate that theinvention also encompasses the use of antigen recognizing immunoglobulinfragments. Such imunoglobulin fragments may include, for example, theFab′, F(ab′)₂, F_(v) or Fab fragments, or other antigen recognizingimmunoglobulin fragments. Such immunoglobulin fragments can be prepared,for example, by proteolytic enzyme digestion, for example, by pepsin orpapain digestion, reductive alkylation, or recombinant techniques. Thematerials and methods for preparing such immunoglobulin fragments arewell-known to those skilled in the art. See generally, Parham, J.Immunology, 131, 2895 (1983); Lamoyi et al., J. Immunological Methods,56, 235 (1983); Parham, Id., 53, 133 (1982); and Matthew et al., Id.,50, 239 (1982).

[0262] The immunoglobulin can be a “chimeric antibody” as that term isrecognized in the art. Also, the immunoglobulin may be a “bifunctional”or “hybrid” antibody, that is, an antibody which may have one arm havinga specificity for one antigenic site, such as a tumor associated antigenwhile the other arm recognizes a different target, for example, a haptenwhich is, or to which is bound, an agent lethal to the antigen-bearingtumor cell. Alternatively, the bifunctional antibody may be one in whicheach arm has specificity for a different epitope of a tumor associatedantigen of the cell to be therapeutically or biologically modified. Inany case, the hybrid antibodies have a dual specificity, preferably withone or more binding sites specific for the hapten of choice or one ormore binding sites specific for a target antigen, for example, anantigen associated with a tumor, an infectious organism, or otherdisease state.

[0263] Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA 0 105 360, to which those skilled inthe art are referred. Such hybrid or bifunctional antibodies may bederived, as noted, either biologically, by cell fusion techniques, orchemically, especially with cross-linking agents or disulfidebridge-forming reagents, and may be comprised of whole antibodies and/orfragments thereof. Methods for obtaining such hybrid antibodies aredisclosed, for example, in PCT application W083/03679, published Oct.27, 1983, and published European Application EPA 0 217 577, publishedApr. 8, 1987, both of which are incorporated herein by reference.Particularly preferred bifunctional antibodies are those biologicallyprepared from a “polydoma” or “quadroma” or which are syntheticallyprepared with cross-linking agents such as bis-(maleimido)-methyl ether(“BMME”), or with other cross-linking agents familiar to those skilledin the art.

[0264] In addition the immunoglobin may be a single chain antibody(“SCA”). These may consist of single chain Fv fragments (“scFv”) inwhich the variable light (“V_(L)”) and variable heavy (“V_(H)”) domainsare linked by a peptide bridge or by disulfide bonds. Also, theimmunoglobulin may consist of single V_(H) domains (dAbs) which possessantigen-binding activity. See, e.g., G. Winter and C. Milstein, Nature,349, 295 (1991); R. Glockshuber et al., Biochemistry 29, 1362 (1990);and E. S. Ward et al., Nature 341, 544 (1989).

[0265] Especially preferred for use in the present invention arechimeric monoclonal antibodies, preferably those chimeric antibodieshaving specificity toward a tumor associated antigen. As used herein,the term “chimeric antibody” refers to a monoclonal antibody comprisinga variable region, i.e., binding region, from one source or species andat least a portion of a constant region derived from a different sourceor species, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are especially preferred in certain applications of theinvention, particularly human therapy, because such antibodies arereadily prepared and may be less immunogenic than purely murinemonoclonal antibodies. Such murine/human chimeric antibodies are theproduct of expressed immunoglobulin genes comprising DNA segmentsencoding murine immunoglobulin variable regions and DNA segmentsencoding human immunoglobulin constant regions. Other forms of chimericantibodies encompassed by the invention are those in which the class orsubclass has been modified or changed from that of the originalantibody. Such “chimeric” antibodies are also referred to as“class-switched antibodies.” Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art. See, e.g., Morrison, S. L., et al., Proc.Nat'l Acad. Sci., 81, 6851 (1984).

[0266] Encompassed by the term “chimeric antibody” is the concept of“humanized antibody,” that is those antibodies in which the framework or“complementarity determining regions (“CDR”) have been modified tocomprise the CDR of an immunoglobulin of different specificity ascompared to that of the parent immunoglobulin. In a preferredembodiment, a murine CDR is grafted into the framework region of a humanantibody to prepare the “humanized antibody.” See, e.g., L. Riechmann etal., Nature 332, 323 (1988); M. S. Neuberger et al., Nature 314, 268(1985). Particularly preferred CDR's correspond to those representingsequences recognizing the antigens noted above for the chimeric andbifunctional antibodies. The reader is referred to the teaching of EPA 0239 400 (published Sep. 30, 1987), incorporated herein by reference, forits teaching of CDR modified antibodies.

[0267] One skilled in the art will recognize that abifunctional-chimeric antibody can be prepared which would have thebenefits of lower immunogenicity of the chimeric or humanized antibody,as well as the flexibility, especially for therapeutic treatment, of thebifunctional antibodies described above. Such bifunctional-chimericantibodies can be synthesized, for instance, by chemical synthesis usingcross-linking agents and/or recombinant methods of the type describedabove. In any event, the present invention should not be construed aslimited in scope by any particular method of production of an antibodywhether bifunctional, chimeric, bifunctional-chimeric, humanized, or anantigen-recognizing fragment or derivative thereof.

[0268] In addition, the invention encompasses within its scopeimmunoglobulins (as defined above) or immunoglobulin fragments to whichare fused active proteins, for example, an enzyme of the type disclosedin Neuberger et al., PCT application, WO86/01533, published Mar. 13,1986. The disclosure of such products is incorporated herein byreference.

[0269] As noted, “bifunctional,” “fused,” “chimeric” (includinghumanized), and “bifunctional-chimeric” (including humanized) antibodyconstructions also include, within their individual contextsconstructions comprising antigen recognizing fragments. As one skilledin the art will recognize, such fragments could be prepared bytraditional enzymatic cleavage of intact bifunctional, chimeric,humanized, or chimeric-bifunctional antibodies. If, however, intactantibodies are not susceptible to such cleavage, because of the natureof the construction involved, the noted constructions can be preparedwith immunoglobulin fragments used as the starting materials; or, ifrecombinant techniques are used, the DNA sequences, themselves, can betailored to encode the desired “fragment” which, when expressed, can becombined in vivo or in vitro, by chemical or biological means, toprepare the final desired intact immunoglobulin “fragment.” It is inthis context, therefore, that the term “fragment” is used.

[0270] Furthermore, as noted above, the immunoglobulin (antibody), orfragment thereof, used in the present invention may be polyclonal ormonoclonal in nature. Monoclonal antibodies are the preferredimmunoglobulins, however. The preparation of such polyclonal ormonoclonal antibodies now is well known to those skilled in the art who,of course, are fully capable of producing useful immunoglobulins whichcan be used in the invention. See, e.g., G. Kohler and C. Milstein,Nature 256, 495 (1975). In addition, hybridomas and/or monoclonalantibodies which are produced by such hybridomas and which are useful inthe practice of the present invention are publicly available fromsources such as the American Type Culture Collection (“ATCC”) 12301Parklawn Drive, Rockville, Md. 20852 or, commercially, for example, fromBoehringer-Mannheim Biochemicals, P.O. Box 50816, Indianapolis, Ind.46250.

[0271] Particularly preferred monoclonal antibodies for use in thepresent invention are those which recognize tumor associated antigens.Such monoclonal antibodies, are not to be so limited, however, and mayinclude, for example, the following: Antigen Site Monoclonal RecognizedAntibodies Reference Lung Tumors KS1/4 N. M. Varki et al., Cancer Res.44:681 (1984). 534, F8, F. Cuttitta et al., in: G. L. Wright (ed)Monoclonal 604A9 Antibodies and Cancer, Marcel Dekker, Inc., NY., p.161, 1984. Squamous Lung G1, LuCa2, Kyoizumi et al., Cancer Res. 45:327(1985). LuCA3, LuCA4 Small Cell Lung TFS-2 Okabe et al., Cancer Res.45:1930 (1985). Cancer Colon Cancer 11.285.14 G. G. Rowland et al.,Cancer Immunol. Immunother. 14.95.55 19:1 (1985). NS-3a-22, Z.Steplewski et al., Cancer Res. 41:2723 (1981). NS-10 NS-19-9, NS-33aNS-52a, 17-1A Carcinoembryonic moAb 35 or Acolla, R. S. et al., Proc.Natl. Acad. Sci. (USA) 77:563 ZCE025 (1980). Melanoma 9.2.27 T. F. Bumoland R. A. Reisfeld, Proc. Natl. Acad. Sci. (USA) 79:1245 (1982). p9796.5 K. E. Hellstrom et al., Monoclonal Antibodies and Cancer, loc. cit.p. 31. Antigen T65 T101 Boehringer-Mannheim P.O. Box 50816 Indianapolis,IN 46250 Ferritin Antiferrin Boehringer-Mannheim P.O. Box 50816Indianapolis, IN 46250 R24 W. G. Dippold et al., Proc. Natl. Acad. Sci(USA) 77:6114 (1980). Neuroblastoma P1 153/3 R. H. Kennet and F.Gilbert, Science 203:1120 (1979). MIN 1 J. T. Kemshead in MonoclonalAntibodies and Cancer, loc. cit. p. 49. UJ13A Goldman et al., Pediatrics105:252 (1984). Glioma BF7, GE2, N. de Tribolet et al., in MonoclonalAntibodies and CG12 Cancer, loc. cit. p. 81. Ganglioside L6 I. Hellstromet al., Proc. Natl Acad. Sci. (USA) 83:7059 (1986); U.S. Pat. Nos.4,906,562, issued Mar. 6, 1990 and 4,935,495, issued Jun. 19, 1990.Chimeric L6 U.S. Ser. No. 07/923,244, filed Oct. 27, 1986, equivalent toPCT Patent Publication, WO 88/03145, published May 5, 1988. Lewis Y BR64U.S. Ser. Nos. 07/289,635, filed Dec. 22, 1988, and U.S. Ser. No.07/443,696, filed Nov. 29, 1989, equivalent to European PatentPublication, EP A 0 375 562, published Jun. 27, 1990. fucosylated LewisBR96, U.S. Ser. Nos. 07/374,947, filed Jun. 30, 1989, and Y ChimericU.S. Ser. No. 07/544,246, filed Jun. 26, 1990, BR96 equivalent to PCTPatent Publication, WO 91/00295, published Jan. 10, 1991. Breast CancerB6.2, B72.3 D. Colcher et al., in Monoclonal Antibodies and Cancer, loc.cit. p. 121. Osteogenic 792T/48, M. J. Umbleton, ibid, p. 181. carcoma792T/36 Leukemia CALL 2 C. T. Teng et al., Lancet 1:01 (1982).anti-idiotype R. A. Miller et al., N. Eng. J. Med. 306:517 (1982).Ovarian Cancer OC 125 R. C. Bast et al., J. Clin. Invest. 68:1331(1981). Prostate Cancer D83.21, J. J. Starling et al., in MonoclonalAntibodies and P6.2, Turp- Cancer, loc. cit., p. 253. 27 Renal CancerA6H, D5D P. H. Lang et al., Surgery 98:143 (1985).

[0272] In the most preferred embodiment, the ligand containing conjugateis derived from chimeric antibody BR96, “ChiBR96,” disclosed in U.S.Ser. No. 07/544,246, filed Jun. 26, 1990, and which is equivalent to PCTPublished Application, WO 91,00295, published Jan. 10, 1991. ChiBR96 isan internalizing murine/human chimeric antibody and is reactive, asnoted, with the fucosylated Lewis Y antigen expressed by human carcinomacells such as those derived from breast, lung, colon and ovariancarcinomas. The hybridoma expressing chimeric BR96 and identified asChiBR96 was deposited on May 23, 1990, under the terms of the BudapestTreaty, with the American Type Culture Collection (“ATCC”), 12301Parklawn Drive, Rockville, Md. 20852. Samples of this hybridoma areavailable under the acession number ATCC HB 10460. ChiBR96 is derived,in part, from its source parent, BR96. The hybridoma expressing BR96 wasdeposited, on Feb. 21, 1989, at the ATCC, under the terms of theBudapest Treaty, and is available under the accession number HB 10036.The desired hybridoma is cultured and the resulting antibodies areisolated from the cell culture supernatant using standard techniques nowwell known in the art. See, e.g., “Monoclonal Hybridoma Antibodies:Techniques and Applications,” Hurell (ed.) (CRC Press, 1982).

[0273] In another highly preferred embodiment the immunoconjugate isderived from the BR64 murine monoclonal antibody disclosed in U.S. Ser.Nos. 07/289,635, filed Dec. 22, 1988, and 07/443,696, filed Nov. 29,1989, equivalent to European Published Application EP A0 375 562,published Jun. 27, 1990. As noted above, this antibody also isinternalizing and is reactive with the Lewis Y antigen expressed bycarcinoma cells derived from the human colon, breast, ovary and lung.The hybridoma expressing antibody BR64 and is identified as BR64 wasdeposited on Nov. 3, 1988, under the terms of the Budapest Treaty, withthe ATCC and is available under the accession number HB 9895. Thehybridoma is cultured and the desired antibody is isolated usingstandard technqiues well known in the art, such as those referencedabove.

[0274] In a third highly preferred embodiment, an immunoconjugate of theinvention is derived from the L6 murine monoclonal antibody disclosed inU.S. Pat. Nos. 4,906,562, issued Mar. 6, 1990, and 4,935,495, issuedJun. 19, 1990. L6 is a non-internalizing antibody active against aganglioside antigen expressed by human carcinoma cells derived formhuman non-small cell lung, breast, colon or ovarian carcinomas. Thehybridoma expressing L6 and identified as L6 was deposited under theterms of the Budapest Treaty on Dec. 6, 1984 at the ATCC and isavailable under the accession number HB 8677. The hybridoma is culturedand the desired antibody is isolated using the standard techniquesreferenced above. A chimeric form of the L6 antibody, if desired, isdescribed in U.S. Ser. No. 07/923,244, equivalent to PCT PublishedApplicaiton, WO 88/03145, published May 5, 1988.

[0275] Thus, as used “immunoglobulin” or “antibody” encompasses withinits meaning all of the immunogloblin/antibody forms or constructionsnoted above.

[0276] The Intermediates and the Conjugates

[0277] The invention provides as intermediates a Michael AdditionReceptor- and acylhydrazone-containing drug derivative of Formula (IIa):

[0278] in which D is a drug moiety, n is an integer from 1 to 10 and Ris a Michael Addition Receptor, all of which are as defined above.

[0279] An especially preferred intermediate encompassed by Formula (IIa)and which is useful for preparation of a conjugate of the invention isone defined by Formula (IIb):

[0280] in which

[0281] R1 is —CH₃, —CH₂OH, —CH₂OCO(CH₂)₃CH₃ or —CH₂OCOCH(OC₂H₅)₂;

[0282] P₃ is —OCH₃, —OH or hydrogen;

[0283] R₄ is —NH₂, —NHCOCF₃, 4-morpholinyl, 3-cyano-4-morpholinyl,1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine,cyanomethyl amine or 1-cyano-2-methoxyethyl amine.

[0284] R₅ is —OH, —OTHP or hydrogen;

[0285] R₆ is —OH or hydrogen, provided that R₆ is not —OH when R₅ is —OHor —OTHP.

[0286] N is an integer from 1 to 10; and,

[0287] R is a Michael Addition receptor moiety.

[0288] The most preferred intermediate for use in the present inventionis defined by Formula (IIc):

[0289] in which R₁, R₃, R₄, R₅ and R₆ are as defined above for Formula(IIb).

[0290] Also used as an intermediate in the invention is a targetingligand which contains a freely reactive sulfhydryl group. The sulfhydrulgroup can be contained within the native targeting ligand or can bederived directly from the ligand or from a derivatized form of theligand. In the preferred method for preparing the conjugates of theinvention, a sulfhydryl group on the ligand or modified ligand reactsdirectly with the Michael Addition Receptor of intermediate of Formula(IIa) to form the final conjugate. Using this process, generally betweenabout one and about ten drug molecules may be linked to each ligand.Thus, in Formula (I), g may be from about 1 to about 10.

[0291] When the conjugate is formed, the Michael Addition Receptorportion becomes a “Michael Addition Adduct,” as used herein. Thus, forexample, as one skilled in the art will appreciate, if the MichaelAddition receptor moiety in the Formulae (IIa) or (IIb) compound is amaleimido moiety, the corresponding “Michael Addition Adduct” portion ofthe final conjugate of Formula (I) will be a succinimido moiety. Thus, a“Michael Addition Adduct” refers to a moiety which would be obtained hada Michael Addition Receptor, as defined in more detail below, undergonea Michael Addition reaction.

[0292] One skilled in the art understands that in the synthesis ofcompounds of the invention, one may need to protect or block variousreactive functionalities on the starting compound and intermediateswhile a desired reaction is carried out on other portions of themolecule. After the desired reactions are complete, or at any desiredtime, normally such protecting groups will be removed by, for example,hydrolytic or hydrogenolytic means. Such protection and deprotectionsteps are conventional in organic chemistry. One skilled in the art isreferred to Protective Groups in Organic Chemistry, McOmie, ed., PlenumPress, N.Y., N.Y. (1973); and Protective Groups in Organic Synthesis,Greene, ed., John Wiley & Sons, New York, N.Y., (1981) for the teachingof protective groups which may be useful in the preparation of compoundsof the present invention.

[0293] By way of example only, useful amino-protecting groups mayinclude, for example, C₁-C₁₀ alkanoyl groups such as formyl, acetyl,dichloroacetyl, propionyl, hexanoyl, 3,3-diethylhexanoyl,γ-chlorobutyryl, and the like; C₁-C₁₀ alkoxycarbonyl and C₅-C₁₅aryloxy-carbonyl groups such as tert-butoxycarbonyl, benzyloxycarbonyl,allyloxycarbonyl, 4-nitro-benzyloxycarbonyl and cinnamoyloxycarbonyl,halo-(C₂-C₁₀)-alkoxycarbonyl such as 2,2,2-trichloroethoxy-carbonyl; andC₁-C₁₅ arylalkyl and alkenyl groups such as benzyl, phenethyl, allyl,trityl, and the like. Other commonly used amino-protecting groups arethose in the form of enamines prepared with β-keto-esters such as methylor ethyl acetoacetate.

[0294] Useful carboxy-protecting groups may include, for example, C₁-C₁₀alkyl groups such as methyl, tert-butyl, decyl; halo-C₁-C₁₀ alkyl suchas 2,2,2-trichloroethyl, and 2-iodoethyl; C₅-C₁₅ arylalkyl such asbenzyl, 4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenylmethyl,C₁-C₁₀ alkanoyloxymethyl such as acetoxymethyl, propionoxymethyl and thelike; and groups such as phenacyl, 4-halophenacyl, allyl, dimethylallyl,tri-(C₁-C₃ alkyl)silyl, such as trimethylsilyl,β-p-toluenesulfonylethyl, β-p-nitrophenyl-thioethyl,2,4,6-triomethylbenzyl, β-methylthioethyl, pthalimidomethyl,2,4-dinitrophenylsulphenyl, 2-nitrobenzhydryl and related groups.

[0295] Similarly, useful hydroxy protecting groups may include, forexample, the formyl group, the chloroacetyl group, the benzyl group, thebenzhydryl group, the trityl group, the 4-nitrobenzyl group, thetrimethylsilyl group, the phenacyl group, the tert-butyl group, themethoxymethyl group, the tetrahydropyranyl group, and the like.

[0296] In general, the intermediate Michael Addition Receptor containinghydrazone drug derivative of Formulae (IIa), (IIb), or (IIc) may beprepared, depending on the Michael Addition Receptor moiety used, byreaction of the drug (or derivatized drug) with a hydrazide containing aMichael Addition Receptor in the general manner described in Method A:

[0297] As noted below, Method A is the preferred method when the MichaelAddition Receptor is a maleimido moiety.

[0298] Alternatively, the Formula (IIa) compound may be prepared byreaction of the drug with a hydrazide to form an intermediate hydrazonedrug derivative followed by reaction of this compound with a MichaelAddition Receptor containing moiety according to the general processdescribed in Method B:

[0299] In Method A and Method B, D, n and R have the meanings previouslynoted. In Method B, L represents a leaving group, such as for example,halogen, mesylate or tosylate, capable of undergoing nucleophilicdisplacement while C represents a group which renders the MichaelAddition Receptor, R, a good nucleophilic reagent. Particularly usefulgroups represented by C may include, for example, alkali metal ions suchas Na⁺, K⁺ or Li⁺.

[0300] A “Michael Addition Receptor,” as one skilled in the art willunderstand, is a moiety capable of reacting with a nucleophilic reagentso as to undergo a nucleophilic addition reaction characteristic of aMichael Addition reaction. As noted, after the nucleophilic additionoccurs, the Michael Addition Receptor moiety is referred to as a“Michael Addition Adduct.”

[0301] Michael Addition Receptors generally used in the Method A processmay include, for example, α,β-ethylenic acids or α,β-thioacids such asthose containing a —C═C—COOH, —C═C—C(O)SH, —C═C—C(S)SH, or a —C═C—C(S)OHmoiety; α,β-ethylenic esters or thio-esters where the alkyl moiety isother than methyl or ethyl, for example, those which contain a—C═C—COOR, —C═C—C(S)OR, —C═C—C(S)SR, or —C═C—C(O)—SR moiety, wherein Ris an ester forming group other than methyl or ethyl; α,β-ethylenicamides, imides, thioamides and thioimides (whether cyclic or acylic),for example, those which contain a moiety such as —C═C—CONR₂,—C═C—CONHCO—, —C═C—CSNR₂, —C═C—CSNHCO—, or —C═C—CSNHCS—, whether cyclicor acyclic and in which —CONR₂ or —CSNR₂ represents a primary,secondary, or tertiary amide or thioamide moiety; α,β-acetylenic acidsor thioacids, for example, those containing a moiety such as —C≡C—COOH,—C≡C—C(S)OH, —C≡C—C(S)SH, or —C≡C—C(O)—SH; α,β-acetylenic esters, forexample those which contain a moiety such as —C≡C—COOR, —C≡C—C(S)OR,—C≡C—C(S)SR, or —C≡C—C(O)—SR in which R is an ester forming group otherthan methyl or ethyl; α,β-ethylenic nitrites, for example thosecontaining a moiety such as —C═C—C≡N; Michael Addition reactivecyclopropane derivatives, for example, 1-cyano-1-ethoxycarbonylcyclopropane

[0302] a vinyl dimethyl-sulphonium bromide, for example, one containinga —C═C—S⁺(Me)₂Br⁻ moiety; an α,β-ethylenic sulfone, for example, onecontaining a

[0303] moiety; α,β-ethylenic nitro compounds, for example, onecontaining —C═C—NO₂ moiety; α,β-ethylenic phosphonium compounds, forexample one containing a

[0304] group; a compound containing a grouping such as C═C—C═N, as wouldbe found, for example, in an aromatic heterocyle such as a 2- or 4-vinylpyridine; or a compound containing an α,β-unsaturated thionium ionmoiety, such as

[0305] Michael Addition Receptors used in Method B may includeα,β-ethylenic aldehydes, for example those compounds containing a—C═C—CHO moiety; α,β-ethylenic ketones, for example those compoundscontaining a

[0306] moiety; α,β-ethylenic esters or thio-esters such as compoundscontaining a —C═C—COOR, —C═C—C(S)OR, —C═C—C(S)SR, or —C═C—C(O)—SR moietyin which R is a ester-forming moiety which is methyl or ethyl, e.g.,

[0307] α,β-acetylenic aldehydes or ketones, for example compoundscontaining a —C≡C—CHO or —C≡C—CO— moiety; α,β-acetylenic esters orthio-esters that have methyl or ethyl as their alkyl moiety, for examplea compound containing a —C≡C—COOR, —C≡C—C(S)OR, —C≡C—C(O)SR or —C≡C—CSSRgroup in which R is an ester forming moiety which is methyl or ethyl.

[0308] One skilled in the art may be familiar with other MichaelAddition Receptors which may be used in the present invention. For ageneral discussion of the Michael Addition Reaction, the reader isreferred to E. D. Bergman, D. Ginsberg, and R. Pappo, Org. React. 10,179-555 (1959); and, D. A. Oare and C. H. Heathcock, Topics inStereochemistry, Vol. 20, eds., E. L. Eliel and S. H. Wilen, John Wileyand Sons, Inc. (1991), and references cited therein.

[0309] The precise reaction conditions used to prepare the intermediatesof Formulae (IIa), (IIb), or (IIc) will depend upon the nature of thedrug and the Michael Addition Receptor used in the reaction. The mostpreferred intermediate of the invention is that represented by Formula(IIc), above, in which the drug moiety is an anthracycline drug and theMichael Addition Receptor is a maleimido group. As noted earlier, forthis reaction, Method A, described above, is used. Upon reaction withthe ligand (thiolated, modified or otherwise), the maleimido MichaelAddition Receptor of the intermediate becomes a succinimido group (theMichael Addition Adduct”) in the final conjugate.

[0310] The sulfhydryl containing ligands exist naturally (i.e., theligand has not been modified) or may be produced, for example, (a) bythiolation of the ligand by reaction with a thiolating reagent such asSMCC or N-succinimid-yl-3-(2-pyridyldithio) propionate (“SPDP”) followedby reduction of the product; (b)thiolation of the native ligand byreaction with iminothiolane (“IMT”); (c) addition of a sulfhydrylcontaining amino acid residue, for example, a cysteine residue, to theligand should the ligand, for example, a protein peptide or polypeptide,fail to have a reactive and available sulfhydryl moiety; or, (d)reduction of a disulfide bond in a native molecule using a reducingagent useful for such purposes, for example, dithiothreitol (“DTT”).Method (d) is the most preferred method for production of sulfhydrylgroups in antibody molecules used in the conjugates of the invention.

[0311] If a thiolating reagent such as SPDP or iminothiolane is used toprepare a conjugate of the invention, one skilled in the art willappreciate that a short “spacer” residue will be inserted between theMichael Addition Receptor moiety and the ligand in the conjugate ofFormula (I). In such a case, z will be 1 in the Formula (I) compound. Inthe situation in which a free sulfhydryl group on the ligand is useddirectly, for example by use of a DTT reduced ligand (particularly a“relaxed” antibody prepared using for example, DTT), or in which areactive residue, for example, cysteine is inserted into the ligandportion of the molecule, z in Formula (I) will be 0 and a directthioether bond will exist between the binding ligand and the MichaelAddition portion of the molecule.

[0312] To form a conjugate, the thiolated ligand, or ligand having afreely reactive sulfhydryl group, is reacted with the Michael Additionreceptor containing hydrazone of Formula (IIa). In general, the reactionconditions must be chosen with regard to the stability of the ligand,the drug and the desired number of drug moieties to be linked to theligand. For example, one skilled in the art will appreciate that theaverage number of drug molecules linked to the ligand can be varied by(1) modifying the amount of the intermediate drug-hydrazone of Formula(IIa) relative to the number of reactive sulfhydryl groups on the ligandmoiety of the conjugate; or (2)(a) modifying the number of reactivesulfhydryl groups on the ligand by, for example, only partially reducingthe ligand (in the case of a protein, peptide or polypeptide), (b) byinserting a limited number of, for example, cysteine residues to aprotein, peptide or polypeptide, or (c) by limiting the degree ofthiolation using less than maximal amounts of thiolation agents, forexample, SPDP or iminothiolane. Although the —SH titer can be varied,the preferred level of free sulfhydryl groups, particularly for arelaxed antibody, is the maximum obtainable using the particularreagents in question. The degree of variation in the —SH titer is easilycontrolled in the relaxed antibody process. For example, FIG. 62 showsthe effect on —SH titer for antibodies BR64 and chimeric BR96 dependingon the mole ratio of DTT to ligand, at 37° C., for a 1.5 hour reaction.One skilled in the art will appreciate that different classes orsubclasses of immunoglobulins can have different numbers of disulfidebridges susceptible to reduction by reagents such as DTT. Thus, afurther consideration in determining the desired level of conjugation ofan antibody or antibody fragment is the number of disulfide groupsavailable for reduction to free —SH groups. In general, however, thepreferred conjugate of Formula (I) will have, on the average from agiven reaction, from about 1 to about 10 drug molecules per ligandmolecule. An especially preferred average drug to ligand molar ratio(“MR”) is about 4 to about 8.

[0313] After the reaction of the conjugate is complete, the conjugatemay be isolated and purified using commonly known dialysis,chromatographic and/or filtration methods. A final solution containingthe conjugate customarily may be lyophilized to provide the conjugate ina dry, stable form which can be safely stored and shipped. Thelyophilized product eventually can be reconstituted with sterile wateror another suitable diluent for administration. Alternatively, theultimate product may be frozen, for example under liquid nitrogen, andthawed and brought to ambient temperature prior to administration.

[0314] In a first preferred embodiment, the anthracyclic hydrazone ofFormula (IIa) is made by reacting the anthracycline with amaleimido-(C₁-C₁₀)-alkyl hydrazide, or a salt thereof. The reaction isoutlined in Method A, described earlier. The reaction generally iscarried out in two steps. First the maleimido-(C₁-C₁₀)-alkyl hydrazide,or its salt, is prepared. After purification by, for example,chromatography and/or crystallization, either the free base of thehydrazide or the salt are reacted with the desired anthracycline oranthracyline salt. After concentration of the reaction solution, themaleimido-containing hydrazone reaction product of Formula (Ia) iscollected, and if desired, purified by standard purification techniques.

[0315] The Formula (IIa) hydrazone then is reacted with asulfhydryl-containing antibody as described earlier. If the antibody isthiolated using, for example,N-succinimidyl-3-(2-pyridyldithio)propionate (“SPDP”), the thiolationreaction generally is performed in two steps: (1) Reaction of a freeamino group on the antibody with SPDP; and, (2) DTT reduction of theSPDP disulfide to yield a free —SH group. In a preferred procedure, inStep (1) of the thiolation reaction, the SPDP/antibody molar ratioranges between about 7.5:1 to about 60:1, depending upon the number ofsulfhydryl groups desired, with a preferred range of about 7.5:1 toabout 30:1, especially for BR64, and preferably about 20:1 for BR96. Thereaction is carried out between about 0° C. and about 50° C., with amost preferred temperature of about 30° C. The reaction may be carriedout at a pH range of between about 6 and about 8 with the most preferredpH being about 7.4. The reduction in Step (2), using preferably DTT, isperformed using a DTT/SPDP molar ratio of between about 2.5:1 to about10:1. The most preferred DTT/SPDP molar ratio is about 5:1 and thenumber of moles of SPDP is that which is added in Step (1) of thereaction. The reaction generally is carried out at about 0° C. to about40° C., preferably 0° C. and is usually complete after about 20 minutes.After dialysis and concentration of the solution of thiolated ligand (anantibody in the most preferred embodiment), the molar concentration ofsulfhydryl groups on the ligand is determined and the thiolated ligandis reacted with the desired molar ratio of the hydrazone derivative ofFormula (IIa) relative to the molar amount of reactive sulfhydryl groupson the ligand. Preferably, the ratio is at least about 1:1. Thisreaction generally is performed at a temperature of about 0° C. to about25° C., preferably about 4° C. The resulting conjugate then may bepurified by standard methods. This reaction scheme is outlined in FIGS.49a and 49 b.

[0316] In a second preferred embodiment, the hydrazone of Formula (IIa)is made as described above. The hydrazone then is reacted, as outlinedin FIG. 49c, with an antibody which previously has been thiolated withiminothiolane (“IMT”). Thiolation of the ligand (preferably an antibody)with IMT generally is a one step reaction. The IMT/antibody ratio mayrange from between about 30:1 to about 80:1, preferably about 50:1. Thereaction is performed for about 30 minutes to about 2 hours, preferablyabout 30 minutes, at a pH of about 7 to about 9.5, preferably at a pH ofabout 9, at a temperature of about 20° C. to about 40° C., preferablyabout 30° C. The reaction product then is reacted with the hydrazone ofFormula (IIa) at a temperature of about 0° C. to about 25° C.,preferably at about 4° C. and at a pH of about 7 to about 9.5,preferably about 7.4. The conjugate then is purified using methodsstandard in the art, for example, dialysis, filtration, orchromatography.

[0317] In a third especially preferred embodiment the intermediatehydrazone of Formula (IIa) is made as described above. The hydrazonethen is reacted with a ligand, most preferably, an antibody, in which atleast one disulfide group has been reduced to form at least onesulfhydryl group. An especially preferred ligand is a “relaxedantibody,” as described below. The preferred reducing agent forpreparing a free sulfhydryl group is DTT although one skilled in the artwill understand that other reducing agents may be suitable for thispurpose.

[0318] A “relaxed” antibody, is one in which one or more, or preferably,three or more, disulfide bridges have been reduced. Most preferably, arelaxed antibody is one in which at least four disulfide bridges havebeen reduced. In a preferred process for preparing a relaxed (i.e.,reduced) antibody, the reduction, especially with DTT, and thepurification of the reaction product, is carried out in the absence ofoxygen, under an inert atmosphere, for example, under nitrogen or argon.This process, as described in detail below, allows one to carefullycontrol the degree of reduction. Thus, this process allows one skilledin the art to reproduce at any time the desired level of reduction of aligand and, therefore, the number of free —SH groups available forpreparing a conjugate of the invention.

[0319] In an alternative procedure, the reaction is carried out underambient conditions, however, a sufficiently large amount of the reducingagent, preferably DTT, is used to overcome any reoxidation of thereduced disulfide bonds which may occur. In either case, purification ofthe product, is carried out as soon as possible after the reaction iscomplete and most preferably under an inert atmosphere such as an argonor nitrogen blanket. The preferred method for preparing the freesulfhydryl containing ligand, however, is the process in whichatmospheric oxygen is excluded from the reaction. An antibody producedby either method is referred to as a “relaxed” antibody. The product,however prepared, should be used for subsequent reaction as quickly aspossible or stored under conditions which avoid exposure to oxygen,preferably under an inert atmosphere.

[0320] In the process in which oxygen is excluded from the reaction(i.e., the reaction is performed under an inert atmosphere), the ligandis incubated, for a period of about 30 minutes to about 4 hours,preferably about 3 hours, with a molar excess of DTT. The DTT1/ligandratios may range between about 1:1 to about 20:1, preferably about 1:1to about 10:1, most preferably about 7:1 to about 10:1, depending uponthe number of sulfhydryl groups desired. For a reduction performed inthe presence of oxygen, the mole ratio of DTT to ligand ranges fromabout 50:1 to about 400:1, preferably from about 200:1 to about 300:1.This latter reaction is carried out for about 1 to about 4 hours,preferably 1.5 hours, at a temperature of between about 20° C. and about50° C., with a preferred temperature being about 37° C. The reaction iscarried out at a pH of between about 6 and about 8, preferably betweenabout 7 to 7.5 The product then is purified using standard purificationtechniques such as dialysis, filtration and/or chromatography. Apreferred purification method is diafiltration. To prevent reoxidationof —SH groups, during purification and storage, the product preferablyis maintained under an inert atmosphere to exclude exposure to oxygen.

[0321] One skilled in the art will appreciate that different ligands,particularly an antibody, may possess different degrees ofsusceptibility to reduction and/or reoxidation. Consequently, theconditions for reduction described above may need to be modified inorder to obtain a given reduced ligand such as that described above.Furthermore, alternate means for preparing a reduced antibody useful inthe conjugation process will be evident to one skilled in the art. Thus,however prepared, a reduced ligand used in the preparation of aconjugate of Formula (I) is meant to be encompassed by the presentinvention.

[0322] To prepare a conjugate of Formula (I), as noted earlier, thereduced antibody reaction product is reacted with the hydrazoneintermediate of Formula (IIC). The reaction preferably is performedunder an inert atmosphere at a temperature of about 0° C. to about 10°C., preferably at about 4° C. and at a pH of about 6 to about 8,preferably about 7.4 The immunoconjugate is purified using standardtechniques such as dialysis, filtration, or chromatography.

[0323] In another embodiment of the invention, an anthracycline ofFormula (11) is joined to a ligand to which is added a moiety carrying afree sulfhydryl group. In one such embodiment, the ligand is anon-antibody ligand, for example, bombesin. The sulfhydryl may be, forexample, part of a cysteine residue added to the native bombesinmolecule. The anthracycline is joined through a hydrazone moiety to aMichael Addition Receptor containing moiety which then reacts with themodified bombesin to form a conjugate of Formula (I). The product thenis purified with standard techniques such as dialysis, centrifugation,or chromatography.

PREPARATION 1 2,5-Dihydro-2,5-dioxo-1H-pyrrolo-1-hexanoic Acid Hydrazideand Its Trifluoroacetic Acid Salt (“Maleimidocaproyl Hydrazide”)

[0324] Maleimidocaproic acid (2.11 g, 10 mmol) (See, e.g., D. Rich etal., J. Med. Chem. 18:1004 (1975); and, O. Keller et al., Helv. Chim.Acta. 58:531 (1975)) was dissolved in dry tetrahydrofuran (200 mL). Thesolution was stirred under nitrogen, cooled to 4° C. and treated withN-methylmorpholine (1.01 g, 10 mmol) followed by dropwise addition of asolution of isobutyl chloroformate (1.36 g, 10 mmol) in THF (10 mL).After 5 min a solution of t-butyl carbazate (1.32 g, 10 mmol) in THF (10mL) was added dropwise. The reaction mixture was kept at 4° C. for ahalf hour and at room temperature for 1 hour. The solvent was evaporatedand the residue partitioned between ethyl acetate and water. The organiclayer was washed with dilute HCl solution, water and dilute bicarbonatesolution, dried over anhydrous sodium sulfate and the solventevaporated. The material was purified by flash chromatography using agradient solvent system of methylene chloride:methanol (100:1-2). Theprotected hydrazide was obtained in 70% yield (2.24 g).

[0325] This material (545 mg, 2.4 mmol) was dissolved and stirred intrifluoroacetic acid at 0°-4° C. for 8 min. The acid was removed underhigh vacuum at room temperature. The residue was triturated with eitherto yield a crystalline trifluoroacetic acid salt of maleimidocaproylhydrazide (384 mg, 70%). An analytical sample was prepared bycrystallization from methanol-ether, to prepare the product, mp102°-105° C. The NMR and MS were consistent with structure. Anal:Calc'd. for C₁₀H₁₅N₃O₃.0.8CF₃COOH: C, 44.02; H, 4.99; N, 13.28. Found(duplicate analyses): C, 44.16, 44.13; H, 4.97, 5.00; N, 12.74, 12.75.

[0326] The salt (220 mg) was converted to the free base bychromatography over silica using a methylenechloride:methanol:concentrated NH₄OH (100:5:0.5) solvent system. Thematerial obtained (124 mg, 80%) was crystallized from methylenechloride-ether to prepare a final product, mp 92°-93° C. NMR and MS wereconsistent with the structure. Anal: Calc'd. for C₁₀H₁₅N₃O₃: C, 53.33;H, 6.67; N, 18.67. Found: C, 53.12; H, 6.67; N, 18.44.

PREPARATION 2 Maleimidocaproylhydrazone of Adriamycin

[0327] A mixture of adriamycin hydrochloride (44 mg, 0.075 mmol),maleimidocaproyl hydrazide (23 mg, 0.102 mmol), prepared according tothe procedure outlined in Preparation 1, and 2-3 drops oftrifluoroacetic acid in absolute methanol (25 mL) was stirred for 15hours under nitrogen and protected from light. At the end of this periodno free adriamycin was detected by HPLC (mobile phase 0.01 molarammonium acetate:acetonitrile, (70:30)). The solution was concentratedat room temperature under vacuum to 10 mL and diluted with acetonitrile.The clear solution was concentrated to a small volume, the solid wascollected by centrifugation, and the product was dried under high vacuumto yield the title compound. The NMR was consistent with structure. HighResolution MS, calc'd. for C₃₁H₄₂N₄O₁₃: 751.2827; Found 751.2804.

[0328] The hydrazone also was formed by using adriamycin and thetrifluoroacetic acid salt of the hydrazide. Thus, the salt (40 mg, 0.12mmol), prepared according to the process outlined in Procedure 1, andadriamycin hydrochloride (50 mg, 0.086 mmol) were stirred in methanol(30 mL) for 15 hrs. The solution was concentrated to 2 mL and dilutedwith acetonitrile. The red solid was collected by centrifugation anddried under vacuum. The product (28 mg, 43%) was identical in NMR andTLC to the one described above. High Resolution MS calc'd. forC₃₁H₄₂N₄O₁₃: 751.2827; found 751.2819.

[0329] 5. Expression and Purification of Coding Sequences for BR96sFv-PE40

[0330] The DNA sequences encoding the single-chain immunotoxin may beexpressed in a variety of systems as set forth below. The DNA may beexcised from pBW 7.0 by suitable restriction enzymes and ligated intosuitable prokaryotic or eukaryotic expression vectors for suchexpression.

[0331] To propagate the cloned DNA, the expression plasmid pBW 7.0,encoding the single-chain immunotoxin, is first transformed intosuitable host cells, such as the bacterial cell line E. coli strain BL21(lambdaDE3) (provided by Dr. Studier, Brookhaven National Laboratories,New York, described by Chaudhary et al., Proc. Natl. Acad. Sci. USA84:4538-4542 (1987)) using standard procedures appropriate to suchcells. The treatment employing calcium chloride, as described by Cohen,Proc. Natl. Acad. Sci. USA 69:2110 (1972) or the CaCl₂ method describedin Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, 2ndEdition, Cold Spring Harbor Press, (1989), may be used for prokaryotesor other cells which contain substantial cell wall barriers.

[0332] Depending on the host cell used, transformation or transfectionis performed using standard techniques appropriate to such cells. Forexample, transfection into mammalian cells is accomplished usingDEAE-dextran mediated transfection, CaPO₄ co-precipitation, lipofection,electroporation, or protoplast fusion, and other methods known in theart including: lysozyme fusion or erythrocyte fusion, scraping, directuptake, osmotic or sucrose shock, direct microinjection, indirectmicroinjection such as via erythrocyte-mediated techniques, and/or bysubjecting host cells to electric currents. The above list oftransfection techniques is not considered to be exhaustive, as otherprocedures for introducing genetic information into cells will no doubtbe developed.

[0333] Expression in prokaryotic cells is preferred. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta-lactamase (penicillinase) and lactose (lac) promoter systems(Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambdaderived P_(L) promoter and N-gene ribosome binding site (Shimatake etal., Nature 292:128 (1981)).

[0334] Expression of the single-chain immunotoxin is detected byCoomassie stained SDS-PAGE and immunoblotting using both anti-idiotypicantibodies that bind to BR96, and anti-PE antibodies to bind to thePE40-portion of the fusion protein.

[0335] 6. Recovery of Products

[0336] The recombinant immunotoxin may be produced along with a signalsequence in cells capable of processing this sequence for secretion.When secreted into the medium, the immunotoxin is recovered usingstandard protein purification techniques such as anion-exchange andgel-filtration chromatography. Purification may also be performed usingantibodies reactive with the anti-immunoglobulin portion of theimmunotoxin. However, while the procedures are more laborious, it iswithin the means known in the art to purify the molecule from sonicatesor lysates of cells in which it is produced intracellularly in fused ormature form.

[0337] In the preferred embodiment described herein, BR96 sFV-PE40 waspurified using anion-exchange and gel-filtration chromatographies withfast protein liquid chromatography (FPLC) as described by Siegall etal., Proc. Natl. Acad. Sci. USA 85:9738-9742 (1988).

[0338] 7. Uses

[0339] The BR96 antibody of the invention is useful for diagnosticapplications, both in vitro and in vivo, for the detection of humancarcinomas that possess the antigen for which the antibodies arespecific. In vitro diagnostic methods include immunohistologicaldetection of tumor cells (e.g., on human tissue, cells or excised tumorspecimens) or serologic detection of tumor-associated antigens (e.g., inblood samples or other biological fluids).

[0340] Immunohistochemical techniques involve staining a biologicalspecimen such as a tissue specimen with the BR96 antibody of theinvention and then detecting the presence on the specimen of theantibody complexed to its antigen. The formation of suchantibody-antigen complexes with the specimen indicates the presence ofcarcinoma cells in the tissue. Detection of the antibody on the specimencan be accomplished using techniques known in the art such asimmunoenzymatic techniques, e.g., the immunoperoxidase stainingtechnique or the avidin-biotin (ABC) technique, or immunofluorescencetechniques (see, e.g., Ciocca et al., “Immunohistochemical TechniquesUsing Monoclonal Antibodies,” Meth. Enzymol. 121:562-79 (1986);Hellstrom et al., “Monoclonal Mouse Antibodies Raised Against Human LungCarcinoma,” Cancer Research 46:3917-23 (1986); and Kimball (ed.),Introduction To Immunology (2nd Ed.), pp. 113-117 (Macmillan Pub. Co.1986)). For example, immunoperoxidase staining was used as described inExample 2, infra, to demonstrate the reactivity of the BR96 antibodywith lung, breast, colon, and ovary carcinomas and the low reactivity ofthe antibody with normal human tissue specimens.

[0341] 8. Diagnostic Techniques

[0342] Serologic diagnostic techniques involve the detection andquantitation of tumor-associated antigens that have been secreted or“shed” into the serum or other biological fluids of patients thought tobe suffering from carcinoma. Such antigens can be detected in the bodyfluids using techniques known in the art such as radioimmunoassays (RIA)or enzyme-linked immunosorbent assays (ELISA) wherein an antibodyreactive with the “shed” antigen is used to detect the presence of theantigen in a fluid sample (see, e.g., Uotila et al., “Two-Site SandwichELISA With Monoclonal Antibodies To Human AFP,” J. Immunol. Methods42:11 (1981) and Allum et al., supra at pp. 48-51). These assays, usingthe BR96 antibodies disclosed herein, can therefore be used for thedetection in biological fluids of the antigen with which the BR96antibodies react and thus the detection of human carcinoma in patients.Thus, it is apparent from the foregoing that the BR96 antibodies of theinvention can be used in most assays involving antigen-antibodyreactions. These assays include, but are not limited to, standard RIAtechniques, both liquid and solid phase, as well as ELISA assays,immunofluorescence techniques, and other immunocytochemical assays (see,e.g., Sikora et al. (eds.), Monoclonal Antibodies, pp. 32-52 (BlackwellScientific Publications 1984)).

[0343] The invention also encompasses diagnostic kits for carrying outthe assays described above. In one embodiment, the diagnostic kitcomprises the BR96 monoclonal antibody, fragments thereof, fusionproteins or chimeric antibody of the invention, and a conjugatecomprising a specific binding partner for the BR96 antibody and a labelcapable of producing a detectable signal. The reagents can also includeancillary agents such as buffering agents and protein stabilizing agents(e.g., polysaccharides). The diagnostic kit can further comprise, wherenecessary, other components of the signal-producing system includingagents for reducing background interference, control reagents or anapparatus or container for conducting the test. In another embodiment,the diagnostic kit comprises a conjugate of the BR96 antibodies of theinvention and a label capable of producing a detectable signal.Ancillary agents as mentioned above can also be present.

[0344] The BR96 antibody of the invention is also useful for in vivodiagnostic applications for the detection of human carcinomas. One suchapproach involves the detection of tumors in vivo by tumor imagingtechniques. According to this approach, the BR96 antibody is labeledwith an appropriate imaging reagent that produces a detectable signal.Examples of imaging reagents that can be used include, but are notlimited to, radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I, ^(99m)Tc, ³²P, ¹²⁵I,³H, and ¹⁴C, fluorescent labels such as fluorescein and rhodamine, andchemiluminescers such as luciferin. The antibody can be labeled withsuch reagents using techniques known in the art. For example, see Wenseland Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, NewYork (1983) for techniques relating to the radiolabeling of antibodies(see also, Colcher et al., “Use Of Monoclonal Antibodies AsRadiopharmaceuticals For The Localization Of Human Carcinoma XenograftsIn Athymic Mice,” Meth. Enzymol., 121:802-16 (1986)).

[0345] In the case of radiolabeled antibody, the antibody isadministered to the patient, localizes to the tumor bearing the antigenwith which the antibody reacts, and is detected or “imaged” in vivousing known techniques such as radionuclear scanning using, e.g., agamma camera or emission tomography (see, e.g., Bradwell et al.,“Developments In Antibody Imaging,” in Monoclonal Antibodies For CancerDetection and Therapy, Baldwin et al. (eds.), pp. 65-85 (Academic Press1985)). The antibody is administered to the patient in apharmaceutically acceptable carrier such as water, saline, Ringer'ssolution, Hank's solution or nonaqueous carriers such as fixed oils. Thecarrier may also contain substances that enhance isotonicity andchemical stability of the antibody such as buffers or preservatives. Theantibody formulation is administered, for example, intravenously, at adosage sufficient to provide enough gamma emission to allowvisualization of the tumor target site. Sufficient time should beallowed between administration of the antibody and detection to allowfor localization to the tumor target. For a general discussion of tumorimaging, see Allum et al., supra at pp. 51-55.

[0346] 9. Therapeutic Applications of the Antibodies of the Inventionand Fragments Thereof

[0347] The properties of the BR96 antibody: a) very high specificity fortumor cells; b) internalization; c) toxicity to antigen-positive tumorcells alone, i.e., in unmodified form, when used at appropriateconcentrations; and d) complement-dependent cytotoxicity andantibody-dependent cellular cytotoxicity activity, suggest a number ofin vivo therapeutic applications. First, the BR96 antibody can be usedalone to target and kill tumor cells in vivo.

[0348] The antibody can also be used in conjunction with an appropriatetherapeutic agent to treat human carcinoma. For example, the antibodycan be used in combination with standard or conventional treatmentmethods such as chemotherapy, radiation therapy or can be conjugated orlinked to a therapeutic drug, or toxin, as well as to a lymphokine or atumor-inhibitory growth factor, for delivery of the therapeutic agent tothe site of the carcinoma.

[0349] Techniques for conjugating such therapeutic agents to antibodiesare well known (see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy,” in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates,” Immunol. Rev., 62:119-58(1982)).

[0350] The BR96 antibody of the invention is particularly suited for usein a therapeutic conjugate because it is readily internalized within thecarcinoma cells to which it binds and thus can deliver the therapeuticagent to intracellular sites of action.

[0351] Alternatively, the BR96 antibody can be coupled to high-energyradiation, e.g., a radioisotope such as ¹³¹I;, which, when localized atthe tumor site, results in a killing of several cell diameters (see,e.g., Order, “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985)). According to yet anotherembodiment, the BR96 antibody can be conjugated to a second antibody toform an antibody heteroconjugate for the treatment of tumor cells asdescribed by Segal in U.S. Pat. No. 4,676,980.

[0352] Still other therapeutic applications for the BR96 antibody of theinvention include conjugation or linkage, e.g., by recombinant DNAtechniques, to an enzyme capable of converting a prodrug into acytotoxic drug and the use of that antibody-enzyme conjugate incombination with the prodrug to convert the prodrug to a cytotoxic agentat the tumor site (see, e.g., Senter et al., “Anti-Tumor Effects OfAntibody-alkaline Phosphatase,” Proc. Natl. Acad. Sci. USA, 85:4842-46(1988); “Enhancement of the in vitro and in vivo Antitumor Activities ofPhosphorylated Mitomycin C and Etoposide Derivatives by MonoclonalAntibody-Alkaline Phosphatase Conjugates,” Cancer Research 49:5789-5792(1989); and Senter, “Activation of Prodrugs by Antibody-EnzymeConjugates: A New Approach to Cancer Therapy,” FASEB J. 4:188-193(1990)).

[0353] Still another therapeutic use for the BR96 antibody involves use,either in the presence of complement or as part of an antibody-drug orantibody-toxin conjugate, to remove tumor cells from the bone marrow ofcancer patients. According to this approach, autologous bone marrow maybe purged ex vivo by treatment with the antibody and the marrow infusedback into the patient (see, e.g., Ramsay et al., “Bone Marrow PurgingUsing Monoclonal Antibodies,” J. Clin. Immunol. 8(2):81-88 (1988)).

[0354] Furthermore, chimeric BR96, recombinant immunotoxins and otherrecombinant constructs of the invention containing the specificity ofthe antigen-binding region of the BR96 monoclonal antibody, as describedearlier, may be used therapeutically. For example, the single-chainimmunotoxin of the invention, BR96 sFv-PE40 may be used to treat humancarcinoma in vivo.

[0355] Similarly, a fusion protein comprising at least theantigen-binding region of the BR96 antibody joined to at least afunctionally active portion of a second protein having anti-tumoractivity, e.g., a lymphokine or oncostatin can be used to treat humancarcinoma in vivo. Furthermore, recombinant techniques known in the artcan be used to construct bispecific antibodies wherein one of thebinding specificities of the antibody is that of BR96 (see, e.g., U.S.Pat. No. 4,474,893), while the other binding specificity of the antibodyis that of a molecule other than BR96.

[0356] Finally, anti-idiotypic antibodies of the BR96 antibody may beused therapeutically in active tumor immunization and tumor therapy(see, e.g., Hellstrom et al., “Immunological Approaches To TumorTherapy: Monoclonal Antibodies, Tumor Vaccines, And Anti-Idiotypes,” inCovalently Modified Antigens And Antibodies In Diagnosis And Therapy,supra at pp. 35-41).

[0357] The present invention provides a method for selectively killingtumor cells expressing the antigen that specifically binds to the BR96monoclonal antibody or functional equivalent. This method comprisesreacting the immunoconjugate (e.g., the immunotoxin) of the inventionwith said tumor cells. These tumor cells may be from a human carcinoma.

[0358] Additionally, this invention provides a method of treatingcarcinomas (for example human carcinomas) in vivo. This method comprisesadministering to a subject a pharmaceutically effective amount of acomposition containing at least one of the immunoconjugates (e.g., theimmunotoxin) of the invention.

[0359] In accordance with the practice of this invention, the subjectmay be a human, equine, porcine, bovine, murine, canine, feline, andavian subjects. Other warm blooded animals are also included in thisinvention.

[0360] The present invention also provides a method for curing a subjectsuffering from a cancer. The subject may be a human, dog, cat, mouse,rat, rabbit, horse, goat, sheep, cow, chicken. The cancer may beidentified as a retinoblastoma, papillary cystadenocarcinoma of theovary, Wilm's tumor, or small cell lung carcinoma and is generallycharacterized as a group of cells having tumor associated antigens onthe cell surface. This method comprises administering to the subject acancer killing amount of a tumor targeted antibody joined to a cytotoxicagent. Generally, the joining of the tumor targeted antibody with thecytotoxic agent is made under conditions which permit the antibody sojoined to bind its target on the cell surface. By binding its target,the tumor targeted antibody acts directly or indirectly to cause orcontribute to the killing of the cells so bound thereby curing thesubject.

[0361] In accordance with the practice of the invention, the tumortargeted antibody is an internalizing tumor targeted antibody. Examplesinclude BR96, fragments of BR96, and functional equivalents thereof.Functional equivalents of BR96 include any molecule which binds theantigen binding site to which BR96 is directed and is characterized by(1) binding carcinoma cells, (2) internalizing within the carcinomacells to which they bind, and (3) mediating ADCC and CDC effectorfunctions.

[0362] Further, in accordance with the practice of the invention, thetumor targeted antibody may be an internalizing tumor targeted antibodywhich recognizes and binds to the Le^(y) determinant. Although,antibodies directed against the Le^(y) determinant are known, suchantibodies were not known to internalize within the carcinoma cells towhich they bind and/or mediate ADCC and CDC effector functions.

[0363] Further, Le^(y) is a fairly common determinant which isoverexpressed in many cancer and some normal cells. Because its presenceis widely found and thus common in both some tumor and non-tumorigeniccells others have questioned whether such antibodies which recognizeLe^(y) may be therapeutically useful.

[0364] The claimed invention also provides a method of inhibiting theproliferation of mammalian tumor cells. This method comprises contactingthe mammalian tumor cells with a proliferation inhibiting amount (i.e.,effective amount) of a tumor targeted antibody joined to a cytotoxic ortherapeutic agent or anti-tumor drug so as to inhibit proliferation ofthe mammalian tumor cells.

[0365] In one example, the tumor targeted antibody is the monoclonalantibody BR96 produced by hybridoma ATCC HB10036. Other examples includefunctional equivalents of BR96 such as ChiBR96; fragments of BR96;bispecific antibodies with a binding specificity for two differentantigens, one of the antigens being that with which the monoclonalantibody BR96 produced by hybridoma ATCC HB10036 binds; and ahuman/murine recombinant antibody, the antigen-binding region of whichcompetitively inhibits the immunospecific binding of monoclonal antibodyBR96 produced by hybridoma HB10036 to its target antigen.

[0366] Also provided is a method of inhibiting the proliferation ofmammalian tumor cells which comprises contacting the mammalian tumorcells with a sufficient concentration of the immunoconjugate of theinvention so as to inhibit proliferation of the mammalian tumor cells.

[0367] Examples of such immunoconjugates include, but are not limitedto, BR96-PE, PE-BR96 fragment, BR96-RA, BR96 (Fab)-lysPE40, BR96F(ab′)₂-lysPE40, ChiBR96-LysPE40, IL-6-PE40, BR96-DOX.

[0368] The subject invention further provides methods for inhibiting thegrowth of human tumor cells, treating a tumor in a subject, and treatinga proliferative type disease in a subject. These methods compriseadministering to the subject an effective amount of the composition ofthe invention.

[0369] It is apparent therefore that the present invention encompassespharmaceutical compositions, combinations and methods for treating humancarcinomas. For example, the invention includes pharmaceuticalcompositions for use in the treatment of human carcinomas comprising apharmaceutically effective amount of a BR96 antibody and apharmaceutically acceptable carrier.

[0370] The compositions may contain the BR96 antibody or antibodyfragments, either unmodified, conjugated to a therapeutic agent (e.g.,drug, toxin, enzyme or second antibody) or in a recombinant form (e.g.,chimeric BR96, fragments of chimeric BR96, bispecific BR96 orsingle-chain immunotoxin BR96). The compositions may additionallyinclude other antibodies or conjugates for treating carcinomas (e.g., anantibody cocktail).

[0371] The antibody, antibody conjugates and immunotoxin compositions ofthe invention can be administered using conventional modes ofadministration including, but not limited to, intravenous,intraperitoneal, oral, intralymphatic or administration directly intothe tumor. Intravenous administration is preferred.

[0372] The compositions of the invention may be in a variety of dosageforms which include, but are not limited to, liquid solutions orsuspensions, tablets, pills, powders, suppositories, polymericmicrocapsules or microvesicles, liposomes, and injectable or infusiblesolutions. The preferred form depends upon the mode of administrationand the therapeutic application.

[0373] The compositions of the invention also preferably includeconventional pharmaceutically acceptable carriers and adjuvants known inthe art such as human serum albumin, ion exchangers, alumina, lecithin,buffer substances such as phosphates, glycine, sorbic acid, potassiumsorbate, and salts or electrolytes such as protamine sulfate.

[0374] The most effective mode of administration and dosage regimen forthe compositions of this invention depends upon the severity and courseof the disease, the patient's health and response to treatment and thejudgment of the treating physician. Accordingly, the dosages of thecompositions should be titrated to the individual patient. Nevertheless,an effective dose of the compositions of this invention may be in therange of from about 1 to about 2000 mg/m².

[0375] The molecules described herein may be in a variety of dosageforms which include, but are not limited to, liquid solutions orsuspensions, tablets, pills, powders, suppositories, polymericmicrocapsules or microvesicles, liposomes, and injectable or infusiblesolutions. The preferred form depends upon the mode of administrationand the therapeutic application.

[0376] The most effective mode of administration and dosage regimen forthe molecules of the present invention depends upon the location of thetumor being treated, the severity and course of the cancer, thesubject's health and response to treatment and the judgment of thetreating physician. Accordingly, the dosages of the molecules should betitrated to the individual subject.

[0377] The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/m² of surface area is described byFreireich, E. J., et al. Cancer Chemother., Rep. 50(4):219-244 (1966).Adjustments in the dosage regimen may be made to optimize the tumor cellgrowth inhibiting and killing response, e.g., doses may be divided andadministered on a daily basis or the dose reduced proportionallydepending upon the situation (e.g., several divided doses may beadministered daily or proportionally reduced depending on the specifictherapeutic situation).

[0378] It would be clear that the dose of the composition of theinvention required to achieve cures may be further reduced with scheduleoptimization.

[0379] In accordance with the practice of the invention, thepharmaceutical carrier may be a lipid carrier. The lipid carrier may bea phospholipid. Further, the lipid carrier may be a fatty acid. Also,the lipid carrier may be a detergent. As used herein, a detergent is anysubstance that alters the surface tension of a liquid, generallylowering it.

[0380] In one example of the invention, the detergent may be a nonionicdetergent. Examples of nonionic detergents include, but are not limitedto, polysorbate 80 (also known as Tween 80 or (polyoxyethylenesorbitanmonooleate), Brij, and Triton (for example, Triton WR-1339 and TritonA-20).

[0381] Alternatively, the detergent may be an ionic detergent. Anexample of an ionic detergent includes, but is not limited to,alkyltrimethylammonium bromide.

[0382] Additionally, in accordance with the invention, the lipid carriermay be a liposome. As used in this application, a “liposome” is anymembrane bound vesicle which contains any molecules of the invention orcombinations thereof.

[0383] In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the scope of this invention in anymanner.

[0384] ADVANTAGES OF THE INVENTION: Initial studies with variouspreviously known immunoconjugates have been disappointing particularlywith solid tumors. In our effort to improve antibody based therapy ofcarcinomas, we have developed and examined novel immunoconjugates andthe anti-cancer drug doxorubicin (DOX).

[0385] BR96 is important for several reasons. It can triggerirreversible changes in membrane structure which leads to tumor celldeath, most likely through the loss of osmotic control (J. Garrigues, U.Garrigues, I. Hellstrom, K. E. Hellstrom, Am. J. Pathol. 142, 607(1993)). Further, it is an internalizing MAb that cycles in anondegraded form between the intracellular compartment and the mediumfor extended periods of time. The latter characteristic makes BR96 anattractive candidate for targeting to tumors various agents forselective concentration in antigen positive cells.

[0386] The antigen for BR96 is abundantly expressed (>200,000molecules/cell) on human carcinoma lines. BR96 binds, according toimmunohistology, the majority of human carcinomas of the breast, lungand colon. Although BR96, like essentially all MAbs to human tumors, isnot truly tumor-specific, it offers advantages over most otherantibodies which recognize the Le^(y) determinant (K. Lloyd, G. Larson,N. Stromberg, J. Thurin, K. A. Karlsson, Immunogenetics 17, 537 (1983);P. M. Pour, V. E. Tempero, C. Cordon-Cardo, P. Avner, Cancer Res. 48,5422 (1988); J. Sakamoto et al., ibid. 49, 745 (1989); T. F. Orntoft, H.Wolf, H. Clausen, E. Dabelsteen, S. I. Hakomori, Int. J Cancer 43, 774(1989)).

[0387] BR96 is more tumor selective and the normal tissues to which itbinds primarily comprise differentiated cells of the esophagus, stomach,and intestine as well as acinar cells of the pancreas (I. Hellstrom, H.J. Garrigues, U. Garrigues, K. E. Hellstrom, Cancer Res. 50, 2183(1990)).

[0388] BR96 is rapidly internalized into lysosomes and endosomes afterbinding to cells expressing the antigen (J. Garrigues et al. 1993).

[0389] The antibodies mediate antibody-dependent cellular cytotoxicity“antibody-dependent cellular cytotoxicity,” “complement-mediatedcytotoxicity,” and “complement-dependent cytotoxicity.”The antibodiescan kill antigen-positive tumor cells in the unconjugated form ifpresent at a sufficient concentration. The antibody conjugates andrecombinant immunotoxins are useful as reagents for killing tumor cells.The antibodies are also useful in diagnostic methods, such as thedetection of carcinomas by in vitro or in vivo technology.

EXAMPLE 1 Preparation of the BR96 Monoclonal Antibody

[0390] The BR96 monoclonal antibody of the invention was produced usinghybridoma fusion techniques as described previously by M. Yeh et al.,Proc. Natl. Acad. Sci. USA, (1979), supra and Yeh et al., Int. J. Cancer(1982), supra. Briefly, a three-month old BALB/c mouse was immunizedusing as the immunogen explanted cultured cells from a human breastadenocarcinoma, designated 3396 or H3396 (from adenocarcinoma of thebreast from a patient which had been established in culture atBristol-Myers Squibb Co., Seattle, Wash.). Methods for establishing andmaintaining cell lines from carcinomas isolated from patients are fullydescribed in Yeh et al., Proc. Natl. Acad. Sci. USA 76:2927-2931 (1979).The mouse received injections on five occasions: on the first fouroccasions, the mouse received one intraperitoneal injection and 1subcutaneous injection split between 4 sites on the mouse. On the fifthoccasion, the mouse was given only one intraperitoneal injection. Thetotal number of cells injected on each occasion was approximately 10⁷cells. Three days after the last immunization, the spleen was removedand spleen cells were suspended in RPMI culture medium. The spleen cellswere then fused with P3-x63-Ag8.653 mouse myeloma cells in the presenceof polyethylene glycol (PEG) and the cell suspension grown in microtiterwells in selective HAT medium as described by Yeh et al., supra (seealso Kohler and Milstein, Nature 256:495-97 (1975) and Eur. J. Immunol.6:511-19 (1976)). The mixture was seeded to form low density culturesoriginating from single fused cells or clones.

[0391] The supernatants from these hybridoma cultures were then screenedfor direct binding activity on the breast cancer cell line, 3396, and afibroblast cell line obtained from a skin biopsy using an ELISA assaysimilar to that described by Douillard et al., “Enzyme-LinkedImmunosorbent Assay For Screening Monoclonal Antibody Production UsingEnzyme-Labeled Second Antibody,” Meth. Enzymol. 92:168-74 (1983).

[0392] According to this assay, the antigen (with which the antibodybeing screened for is reactive) is immobilized on microtiter plates andthen incubated with hybridoma supernatants. If a supernatant containsthe desired antibody, the antibody will bind to the immobilized antigenand is detected by addition of an anti-immunoglobulin antibody-enzymeconjugate and a substrate for the enzyme which leads to a measurablechange in optical density. In the present studies, breast cancer cellsor control fibroblast cells were dispensed into a 96-well tissue cultureplate (Costar Cambridge, Mass.) and incubated overnight in a humid 37°C. incubator (5% CO₂). The cells were then fixed with 100 μl of freshlyprepared 1.0% glutaraldehyde to a final well concentration of 0.5% andincubated for 15 min at room temperature, followed by washing threetimes with 1×phosphate buffered saline (PBS). The cells were nextblocked for 30 min with 5% bovine serum albumin (BSA) in PBS and washedagain three times with PBS. The supernatants from the hybridoma cultureswere then added at 100 μl/well, the wells incubated for 1 h at roomtemperature, and the cells washed three times with PBS. Next, goatanti-mouse horseradish peroxidase (Zymed, Calif.) diluted in 0.1% BSAand PBS was added to a concentration of 100 μl/well. The reactionmixture was incubated for either 1 h at room temperature or 30 min at37° C. and the cells were then washed three times with PBS.o-Phenylenediamine (OPD) was then added at 100 μl/well and the platesincubated in the dark at room temperature for 5-45 min. Antibody bindingto the cells was detected by a color change in the wells that occurredwithin 10-20 min. The reaction was stopped by adding 100 μl/well H₂SO₄and the absorbance read in a Dynatech (Alexandria, Va.) Microelisaautoreader at 490 nm.

[0393] It should be noted that this assay can be performed using intactcells or purified soluble antigen or cellular extracts as theimmobilized antigen. When soluble antigen or cell extracts were used asantigen, the antigen was initially plated at 50 μl/well in PBS and theplates were incubated overnight at room temperature before beginning theassay. When using intact cells as antigen, they may be used fresh orafter fixation. In either case, the cells were initially plated at 10⁴cells in 100 μl/well in culture medium and incubated overnight in a 37°C. incubator (5% CO₂).

[0394] Hybridomas which produced antibodies binding to the breast cancercell line and not to the human fibroblast cells were thus selected, andtested in a FACS cell sorter on peripheral blood leukocytes (PBLs), asdescribed in Example 2, infra. Hybridomas that were negative on PBLswere cloned, expanded in vitro, and further tested for antibodyspecificity. Those hybridomas producing antibody reactive with humanbreast cancer were recloned, expanded, and injected into pristane-primed3-month old BALB/c mice, where they grew as ascites tumors.

[0395] Following this procedure, hybridoma cell line BR96 was obtained,cloned and injected into mice to develop as an ascites tumor. Asdisclosed above, the BR96 hybridoma has been deposited with the ATCC.Monoclonal BR96 antibody was purified from ascites by affinitychromatography on immobilized recombinant protein A (Repligen,Cambridge, Mass.). Clarified ascites was diluted with an equal volume ofbinding buffer (1 M potassium phosphate, pH 8) and applied to a proteinA column previously equilibrated with binding buffer. The column wasextensively washed with binding buffer and then the antibody was elutedwith 50 mM phosphoric acid, pH 3. The purified antibody fraction wasneutralized with 1 M Tris, pH 9 and then dialyzed against phosphatebuffered saline. Purified BR96 was finally sterile filtered and storedrefrigerated or frozen.

EXAMPLE 2 Characterization of the BR96 Monoclonal Antibody

[0396] Isotype Determination

[0397] To determine the class of immunoglobulin produced by the BR96hybridoma, the following techniques were utilized:

[0398] (a) Ouchterlony Immunodiffusion

[0399] An aliquot of supernatant of the hybridoma cells was placed intothe center well of a 25% agar plate. Monospecific rabbit anti-mouse Igisotype antibodies (Southern Biotechnology, Birmingham, Ala.) wereplaced in the outer wells and the plate was incubated for 24-28 h atroom temperature. Precipitation lines were then read.

[0400] (b) ELISA Isotyping

[0401] Dynatech Immulon 96-well plates were coated with goat anti-mouseIg antibodies at 1 μg/ml concentration, 50 μl/well in PBS and leftcovered overnight at 4° C. The plates were washed with PBS/Tween 20,0.05% and blocked with medium at 100 μl/well for 1 h at roomtemperature. After washing the plates, supernatants from the BR96hybridoma were added and incubated at room temperature for 1 h. Afterwashing with PBS containing 2% bovine serum albumin (BSA), plates wereincubated at 37° C. for 30 min with monospecific rabbit anti-mouse Igisotype antibodies coupled to peroxidase (Zymed, South San Francisco,Calif.). After further washing, the plates were incubated with 1 mg/mlOPD and 0.03% H₂O₂ in 0.1 M citrate buffer, pH 4.5. Optical density at630 nm was determined on a Dynatec ELISA plate reader.

[0402] Based on these procedures, it was determined that the BR96monoclonal antibody is of the IgG3 isotype.

[0403] Characteristics of the BR96 Monoclonal Antibody

[0404] The BR96 antibody shows a high degree of reactivity with a widerange of carcinomas and displays only limited reactivity with normalcells. This was shown by experiments involving immunohistologicalstudies on frozen tissue sections as well as binding studies usingintact cultured cells.

[0405] Immunohistology

[0406] The peroxidase-antiperoxidase (PAP) technique of L. A.Sternberger as described in Immunochemistry, pp. 104-69 (John Wiley &Sons, New York, 1979) and as modified by J. Garrigues et al., “DetectionOf A Human Melanoma-Associated Antigen, p97, In Histological Sections OfPrimary Human Melanomas,” Int. J. Cancer. 29:511-15 (1982), was used forthe immunohistological studies. The target tissues for these tests wereobtained at surgery and frozen within 4 h of removal using isopentaneprecooled in liquid nitrogen. Tissues were then stored in liquidnitrogen or at −70° C. until used. Frozen sections were prepared, airdried, treated with acetone and dried again (see Garrigues et al.,supra). Sections to be used for histologic evaluation were stained withhematoxylin. To decrease non-specific backgrounds sections werepreincubated with normal human serum diluted ⅕ in PBS (see Garrigues etal., supra). Mouse antibodies, rabbit anti-mouse IgG, and mouse PAP werediluted in a solution of 10% normal human serum and 3% rabbit serum.Rabbit anti-mouse IgG (Sternberger-Meyer Immunochemicals, Inc.,Jarettsville, Md.), was used at a dilution of {fraction (1/50)}. MousePAP complexes (Sternberger-Meyer Immunochemicals, Inc.) containing 2mg/ml of specifically purified PAP was used at a dilution of {fraction(1/80)}.

[0407] The staining procedure consisted of treating serial sections witheither specific antibody, i.e., BR96, or a control antibody for 2.5 h,incubating the sections for 30 min at room temperature with rabbitanti-mouse IgG diluted {fraction (1/50)} and then exposing the sectionsto mouse PAP complexes diluted {fraction (1/80)} for 30 min at roomtemperature. After each treatment with antibody, the slides were washedtwice in PBS.

[0408] The immunohistochemical reaction was developed by adding freshlyprepared 0.5% 3,3′-diaminobenzidine tetrahydrochloride (Sigma ChemicalCo., St. Louis, Mo.) and 0.01% H₂O₂ in 0.05 M Tris buffer, pH 7.6, for 8min (see Hellstrom et al., J. Immunol. 127:157-60 (1981)). Furtherexposure to a 1% OsO₄ solution in distilled water for 20 min intensifiedthe stain. The sections were rinsed with water, dehydrated in alcohol,cleared in xylene, and mounted on slides. Parallel sections were stainedwith hematoxylin.

[0409] The slides were each evaluated under code and coded samples werechecked by an independent investigator. Typical slides were photographedby using differential interference contrast optics (Zeiss-Nomarski). Thedegree of antibody staining was evaluated as 0 (no reactivity), + (a fewweakly positive cells), ++ (at least one third of the cells positive),+++ (most cells positive), ++++ (approximately all cells stronglypositive). Because differences between + and 0 staining were less clearcut than between + and ++ staining, a staining graded as ++ or greaterwas considered “positive.” Both neoplastic and stroma cells wereobserved in tumor samples. The staining recorded is that of the tumorcells because the stroma cells were not stained at all or were stainedmuch more weakly than the tumor cells.

[0410] Table 1 below demonstrates the immunohistological staining ofvarious tumor and normal tissue specimens using the BR96 monoclonalantibody. As the table clearly demonstrates, the BR96 antibody reactswith a wide range of human carcinoma specimens, does not react withsarcoma and displays only infrequent reactivity with melanoma.Furthermore, it shows only limited reactivity with any of the largenumber of normal human tissues tested. The only reactivity detected withnormal cells was binding to a small subpopulation of cells in thetonsils and in the testis, and to acinar cells in the pancreas, and tocertain epithelial cells of the stomach and esophagus. TABLE 1IMMUNOPEROXIDASE STAINING OF HUMAN TUMORS AND NORMAL TISSUE SPECIMENSWITH BR96 MONOCLONAL ANTIBODY NUMBER POSITIVE/ TISSUE TYPE NUMBER TESTEDTumors Lung carcinoma (non-small cell) 14/17 Breast carcinoma 17/19Colon carcinoma 15/18 Ovary carcinoma 4/4 Endometrial carcinoma 2/2Melanoma 2/5 Sarcoma 0/5 Stomach carcinoma 2/2 Pancreatic carcinoma 2/2Esophagus carcinoma 2/2 Cervical carcinoma 2/2 Normal Tissues Lung 0/7Spleen 0/5 Breast 0/2 Colon 0/7 Kidney 0/7 Liver 0/5 Brain 0/2 Heart 0/3Skin 0/2 Thyroid 0/2 Adrenal 0/1 Ovary 0/2 Lymph nodes 0/2 Lymphocytepellet 0/4 Pancreas 2/2 (only acinar cells were positive) Uterus 0/7Retina 0/1 Testis 2/2 (only small sub-population of cells were positive)Tonsil 2/2 (only small sub-population of cells were positive) Stomach2/2 (epithelial cells positive) Esophagus 2/2 (epithelial cellspositive)

[0411] The binding of the BR96 antibody to various cultured cell lineswas also evaluated. Antibody binding to the cell surface of intactcultured cells was identified either by a direct binding assay with¹²⁵I-labeled antibody as described in Brown et al., “QuantitativeAnalysis Of Melanoma-Associated Antigen p97 In Normal And NeoplasticTissues,” Proc. Natl. Acad. Sci. USA, 78:539-43 (1981), or by directimmunofluorescence using a Coulter Epics C fluorescence activated cellsorter (FACS) II (Hellstrom et al., Cancer Res. 46:3917-3923 (1986)).

[0412] For binding analyses using a FACS cell sorter, 2×10⁵ to 1×10⁶cultured cells were aliquoted in 15% fetal bovine serum (FBS) in IMDMmedia (Gibco, NY) to a total volume of 500 μl/tube. The cells werecentrifuged for 1.5 min on a Serofuge and the supernatant removed. 100μl of the BR96 monoclonal antibody at 10 μl/ml was added to each tube,the contents of which was then mixed and incubated on ice for 30 min.The reaction mixture was washed three times with 500 μl of 15% FBS/IMDMby centrifugation for 1.5 min on the Serofuge (tubes were blotted afterthe third wash). Then, 50 μl of optimized FITC-conjugated goatanti-mouse IgG antibody (Tago, Burlingame, Calif.) diluted 1:25 in 15%FBS/IMDM was added to each tube and the reaction mixture was mixed andincubated for 30 min. The wash step was then repeated and after blottingof the tubes, each pellet was resuspended in 200-500 μl of PBS. Eachsample was run on a Coulter Epics C FACS and the mean fluorescenceintensity (MFI) was determined. From the MFI, the linear fluorescentequivalent (LFE) was determined. The LFE of each test sample divided bythe LFE of a negative control gave a ratio between the brightness ofcells stained by specific versus control antibody. The binding data isshown in Table 2 below. TABLE 2 FACS ANALYSIS OF THE BINDING OF BR96 TOVARIOUS TYPES OF SUSPENDED CELLS Ratio Cell line (10 μg/ml) Breastcarcinoma 3396 54 Breast carcinoma MCF-7 38 Breast carcinoma 3630 22Breast carcinoma 3680 22 Lung carcinoma 2987 15 Lung carcinoma 2707 30Lung carcinoma 2964 2 Lung carcinoma 3655-3 18 Colon carcinoma RCA 34Colon carcinoma 3619 22 Colon carcinoma 3347 5 Colon carcinoma HCT116 1Colon carcinoma CB5 27 Colon carcinoma C 30 Colon carcinoma 3600 16Ovary carcinoma 3633-3 11 Melanoma 2669 1 Melanoma 3606 1 Melanoma 36201 T cell lymphoma line CEM 1 T cell lymphoma line MOLT-4 1 B celllymphoma line P3HR1 1 Peripheral blood leukocytes 1

[0413] As Table 2 demonstrates, the BR96 monoclonal antibody reactedwith most breast, lung and colon carcinoma cell lines but did not reactwith melanoma lines or with T or B lymphoma lines nor with normalperipheral blood leukocytes. Scatchard analysis using radiolabeledantibody indicated that the approximate association constant (K_(a)) ofBR96 was calculated to be 3.6×10⁶ antigen sites/cell for the 3396 linewhich binds BR96.

[0414] These data demonstrate that monoclonal antibody BR96 recognizecell surface antigens abundantly expressed (up to 10⁶ molecules/cell) onthe majority of human carcinomas.

EXAMPLE 3 Internalization of the BR96 Monoclonal Antibody withinCarcinoma Cells

[0415] Studies were conducted to measure internalization of the BR96monoclonal antibody within antigen-positive carcinoma cells. Accordingto one procedure, BR96 was conjugated to the ricin A chain toxin to forman immunotoxin, BR96-RA, whose internalization by carcinoma cells wasthen determined. Uptake of the conjugate by the carcinoma cells wasassessed by determining to what extent the tumor cells were killed byricin A chain.

[0416] Conjugation of the antibody to the toxin was carried out asfollows: Deglycosylated ricin-A chain (Inland Labs, Austin, Tex.) (see,also, Blakey et al., Cancer Res., 47:947-952 (1987)) was treated withdithiothreitol (5 mM) prior to gel filtration on G-25 Sephadex usingPBS, pH 7.2 as eluant. This was added in a 2:1 molar ratio to theantibody in PBS, the antibody having been previously modified withN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Pierce, Rockford,Ill.) according to the procedure of Lambert et al., J. Biol. Chem.,260:12035-12041 (1985). Reaction was allowed to proceed for 12-24 h atroom temperature, and the solution was then diluted with 1 volume ofH₂O. Removal of unconjugated antibody was achieved using Blue SepharoseCL-6B (Pharmacia, Uppsala, Sweden) (see Knowles et al., Anal. Biochem.,160:440-443 (1987)).

[0417] The conjugate and excess ricin-A chain were eluted with high salt(10×PBS) and subjected to further purification on Sephacryl-300(Pharmacia) using PBS as eluant. The resulting conjugate was free ofunbound monoclonal antibody or ricin A-chain and consisted mostly of 1:1adducts.

[0418] The internalization of BR96-RA by various carcinoma cell lineswas then measured using a thymidine uptake inhibition assay. Accordingto this assay, the inhibition of ³H-thymidine incorporation into the DNAof the carcinoma cells (i.e., the inhibition of cell proliferation) is ameasure of the cytotoxic effect of BR96-RA on the cells and thus ameasure of the internalization of the immunotoxin within the cell.

[0419] For the assay, carcinoma cells were plated into a 96-wellmicrotiter plate at 1×10⁴ cells/well in 100 μl of IMDM medium with 15%fetal calf serum FCS). The plates were incubated for 12-18 h at 37° C.to let the cells adhere. Then the media was removed. Plates were kept onice. The BR96-RA immunotoxin (100 μl) was then added in log 10 serialdilutions, starting at 10 μg/ml final concentration down to 0.01 μg/ml.The reaction mixture was incubated for 4 h on ice. The plates werewashed and 200 μl/ml media was added and further incubated at 37° C. for18 h. At this point, 50 μl of ³H-thymidine was added at 1 μCi/well andthe plates incubated for 6 h at 37° C. in a 5% CO₂ incubator. The assayplates were then frozen at −70° C. for at least 1 h and thawed in a geldryer for 15 min. The cells were harvested onto glass fiber filters(Filter Strips, No. 240-1, Cambridge Technology) in plasticscintillation vials using a PHD cell harvester. 3 ml of scintillationcounting liquid was added to the vials and the vials were counted on aBeckman LS3891 beta scintillation counter at 1 minute per sample.

[0420] Graphs of the percent inhibition of thymidine incorporation vs.immunotoxin concentration for each cell line tested were plotted and areshown in FIGS. 1-5. In each assay, a control was run. The results of theassay are expressed as a percentage of the ³[H] thymidine incorporatedby untreated control cells.

[0421]FIG. 1 depicts the percent inhibition of thymidine incorporationby cells from the 3396 breast carcinoma cell line caused byinternalization of BR96-RA. Similar results were obtained with the 2707lung carcinoma cell line (FIG. 2) and C colon carcinoma cell line (seeFIG. 4). The BR96-RA was not internalized by HCT 116 cell line (ATCC No.CCL 247), a human colon carcinoma cell line that does not bind BR96 (seeFIG. 3). FIG. 5 shows no internalization of BR96-RA on 3347, a coloncarcinoma cell line to which BR96 does not bind; BR6-RA, on the otherhand, which binds to the 3347 cells, does internalize. This study,therefore, demonstrated not only internalization of the BR96 antibodybut the selectivity of the internalization of the BR96 antibody forantigen positive carcinoma cells.

EXAMPLE 4 Cytotoxicity of Unmodified BR96 Monoclonal Antibody

[0422] Three types of experiments were performed to follow up on theunexpected observation that monoclonal antibody BR96 appeared to becytotoxic by itself (i.e., in unmodified state) when tested in a FACSassay. So as to avoid an effect of complement in serum, all sera usedwere heat inactivated (56° C. for 30 min); in addition, some of theexperiments with FACS analysis (as described below) were performed oncells which were grown in serum-free medium and tested in the absence ofserum.

[0423] First, living suspended cells from a variety of antigen positivecarcinoma lines (3396, 2987, 3619) were treated with monoclonal antibodyBR96. Cells (5×10⁵) were incubated on ice for 30 min with 100 μl of BR96or control monoclonal antibody at a concentration of 60, 30, 15, 7.5 and3.8 μg/ml in culture medium (IMDM, 15% FBS). After washing the cellstwice with culture medium, the cells were suspended in 500 μl medium andstained by adding the dye propidium iodide which stains dead cells(Krishan, Cell Biol. 66:188 (1975); and Yeh, J. Immunol. Methods, 43:269(1981)). Out of a 1 mg/ml stock solution (in 70% alcohol) 5 μl dye wasadded to cell samples, incubated on ice for 15 min, washed once andfinally suspended in 500 μl medium. The cells were evaluated on aCoulter Epics C FACS, with dead cells being identified by their redfluorescence. The analysis was done on a two-parameter display with logforward lightscatter in the horizontal and log red fluorescence in thevertical display. Computations of cell size versus cell viability wereobtained by applying the Coulter Epics C Quadstat program. Tumor cellswhich could bind BR96 as well as tumor cells not binding BR96 werestudied in parallel. The results are shown in FIG. 6. FIG. 6demonstrates that incubation of cells from any of three antigen-positivecarcinomas with BR96 rapidly killed them. Untreated or antigen-negativecells were not killed.

[0424] Second, tumor cells (3396, 3630, 2987, 3619 and HCT 116) wereexposed to BR96 (or the control monoclonal antibody) for 18 h at 37° C.in a 96-well microtiter plate at 3×10³ cells/well in 150 μl of IMDMmedium containing FBS for 66 h after which 50 μl of ³[H]-thymidine wasadded at 1 μCi/well and the plate was incubated for another 6 h at 37°C. Subsequently, it was frozen at −70° C. for at least 1 h and thawed ina gel dryer for 15 min, and the cells harvested onto glass fiberfilters. The tritiated thymidine assay was then performed as describedin the preceding example, except that the cells and antibodies wereincubated at 37° C. FIG. 7 illustrates the results. BR96 caused aninhibition of [³H] thymidine incorporation into antigen-positive celllines, and this effect was dose dependent. The antigen-negative cellline HCT116 was not affected by any concentration of BR96 examined.

[0425] Third, using a modification of a procedure described by Linsleyet al. (Linsley et al., “Identification and characterization of cellularreceptors for growth regulator, Oncostatin M,” J. Biol. Chem.264:4282-4289 (1989)) a growth inhibition assay was performed. Cellsfrom four different cell lines (HCT116, 2987, 3396 and 3630) were seeded(3×10³) in a volume of 0.1 ml of IMDM with 15% fetal bovine serum (FBS)in 96-well microtiter plates and allowed to attach for 3 h at 37° C.Various concentrations of whole BR96 monoclonal were then added in avolume of 0.1 ml, after which incubation at 37° C. was continued for 72h. Subsequently, the culture medium was removed and the cells werestained by crystal violet (0.1% in 20% methanol) for 30 min. and washedthree times with PBS. The bound dye was eluted by the addition of 0.1 mlof a solution of 0.1 M sodium citrate, pH 4.2, in 50% ethanol. Sampleswere assayed in triplicate on an ELISA reader measuring the absorbancein the presence of BR96 with the absorbance in untreated samples. Theresults of this procedure are expressed as percentage inhibition of cellgrowth. FIG. 8 illustrates the results. The results of this assay werein agreement with those presented above for the thymidine incorporationassay (FIG. 7).

EXAMPLE 5 Antibody-Dependent Cellular Cytotoxicity Activity of BR96Antibody

[0426] Determination of antibody-dependent cellular cytotoxicityactivity of BR96 monoclonal antibody was performed as described byHellstrom et al., Proc. Natl. Acad. Sci. (USA) 82:1499-1502 (1985).Briefly, a short-term ⁵¹Cr-release test that measures the release of⁵¹Cr as described by Cerrotini et al., Adv. Immunol. 18:67-132 (1974)was used as evidence of tumor-cell lysis (cytotoxicity). Peripheralblood lymphocytes from healthy human subjects were separated onFicoll-Hypaque (Hellstrom et al., Int. J. Cancer 27:281-285 (1981)) toprovide effector cells equal to 5% natural killer cell reactivityagainst SK-MEL-28 cells (ATCC No. HTB 72); 10⁶ cells were labeled byincubation with 100 μCi (1 Ci=37 Gbq) of ⁵¹Cr for 2 h at 37° C., afterwhich they were washed three times and resuspended in medium. Thelabeled cells were seeded (2×10⁴ cells per well in 20 μl) intoMicrotiter V-bottom plates (Dynatech Laboratories, Alexandria, Va.).Purified antibody BR96 (10 μg/ml, 1 μg/ml, and 0.1 μg/ml) was thenadded, followed by 2×10⁵ lymphocytes per well in 100 μl. The mixtureswere incubated for 2 to 4 h after which the plates were centrifuged at400×g. The supernatants were removed and the radioactivity in 100 μlsamples was measured with a gamma-counter. There were two replicates pergroup; the variation between replicates was less than 10%. Several“criss-cross” experiments were done, in which lung (or colon) carcinomaand melanoma targets were tested in parallel with monoclonal antibodyBR96 and with the antimelanoma monoclonal antibody MG-22 (Hellstrom etal., Proc. Natl. Acad. Sci. USA, 82:1499-1502 (1985)) which do not bindto most carcinoma cells. Controls included the incubation of targetcells alone or with either lymphocytes or monoclonal antibodyseparately.

[0427] Spontaneous release was defined as the counts per minute (cpm)released into the medium from target cells exposed to neither antibodiesnor lymphocytes, and total release, as the number of counts releasedfrom target cells that were osmotically lysed at the end of the assay.Percent cytotoxicity was calculated as:$\frac{\text{experimental~~group~~release} - \text{spontaneous release}}{\text{total~~release} - \text{spontaneous release}} \times 100$

[0428] Effector cells were characterized by assessing their sensitivityto incubation with anti-serum to the Leu-11b surface marker and guineapig complement, using procedures described by Hellstrom et al., inMonoclonal Antibodies and Cancer Therapy, UCLA Symposia on Molecular andCellular Biology, New Series, eds. Reisfeld & Sell, Liss, New York, Vol27, pp. 149-164 (1985), incorporated herein by reference. This was doneto measure the expression of the Leu-11b marker, which characterizesnatural killer (NK) cells and is expressed by lymphocytes mediatingantibody-dependent cellular cytotoxicity against human melanoma cells inthe presence of monoclonal antibody BR96. The cytotoxicity by effectorcells alone (“natural killer effect”) was subtracted from the dataprovided in FIG. 9.

[0429] The results shown in FIG. 9 for an antibody concentration of 10μg/ml indicate that BR96 mediates antibody-dependent cellularcytotoxicity activity if present in sufficient concentrations and if thetarget cells express sufficient concentrations of the epitope. Theantibody-dependent cellular cytotoxicity activity can be seen atantibody concentrations lower than those at which the antibody iscytotoxic by itself (usually around 20 μg/ml). When antibody BR96 wasused alone as a control it produced 0% killing at the concentrationstested and using the ⁵¹Cr assay. Antibody-dependent cellularcytotoxicity activity was only found with BR96 antibody-binding celllines. Thus, cells from five different carcinoma lines, which all boundBR96, were killed via antibody-dependent cellular cytotoxicity atmonoclonal antibody concentrations down to 0.1 μg/ml, while cells from asixth line, 2964, which did not bind BR96, were not killed. Therequirement for antibody binding to obtain antibody-dependent cellularcytotoxicity was further demonstrated by the fact that both of the twocarcinomas which could bind a different antibody, L6 (lines 3619 and2987), were killed by L6 via antibody-dependent cellular cytotoxicity,while the others were not. Under the conditions of the assay, BR96 alonecaused the release of only 1% of the label, even when tested at aconcentration of 10 μg/ml.

EXAMPLE 6 Ability of BR96 to Mediate Complement-Mediated Cytotoxicity(Complement-Dependent Cytotoxicity)

[0430] Tests to evaluate the ability of monoclonal antibody BR96 to killtumor cells in the presence of human serum as a source of complement(complement-mediated cytotoxicity or complement-dependent cytotoxicity)were performed similarly to those for the antibody-dependent cellularcytotoxicity tests described in Example 5, supra, except that 100 μl ofhuman serum from normal human subjects as the source of complementdiluted 1:3 to 1:6 was added per microtest well in place of a suspensionof effector cells.

[0431] As shown in FIG. 10, complement-dependent cytotoxicity againstcells binding BR96 was seen at an antibody concentration of 0.1-5.0μg/ml, while there was no complement-dependent cytotoxicity against theBR96 antigen-negative lines HCT116 and 3347. The 3347 cells could,however, be killed when using the L6 monoclonal antibody, which binds tothese cells. Controls were always included in which BR96 was tested inthe absence of complement. No killing by BR96 alone was detected by the⁵¹Cr release assay. These data show that BR96 gave a cytotoxic effect inthe presence of human serum at concentrations where it is not cytotoxicby itself. (Control antibody gave no complement-dependent cytotoxicity).

EXAMPLE 7 Determination of Reactivity of BR96 to Glycolipids andGlycoproteins

[0432] BR96 antibody was tested for reactivity to a variety ofimmobilized glycolipid antigens having known carbohydrate structures andsynthetic glycoproteins (so called “neoglycoproteins”) using an ELISAassay in which purified glycolipids and glycoproteins and antibody wereused in excess (Dr. John Magnani, Biocarb, Gaithersburg, Md.; Lloyd etal., Immunogenetics 17:537-541 (1983)). Glycolipids were dried frommethanol in microtiter wells at 100 ng/well. Synthetic glycoproteinswere coated on the surface of the wells by incubation of glycoproteindiluted to 200 ng in phosphate buffered saline (PBS), at pH 7.4/well.Purified BR96 was assayed at a concentration of 10 μg/ml in 0.01 MTris-HCl, pH 7.4, containing 1% BSA containing antibodies from asciteswere assayed at a dilution of 1:100 in the same buffer. At these highconcentrations most binding interactions are readily detected.Absorbance values were calculated as the average of duplicate wells. Theresults of this analysis are summarized in FIGS. 11 and 12 showing thatBR96 reacted with Le^(y) and Lex determinant.

[0433] These findings indicate that BR96 can bind to a variant form ofthe Lewis Y (Fuc α1-2Galβ1-4(Fucα1-3)GlcNAc) antigen and that fucoseα1-3 attached to GlcNAc forms a portion of the Le^(y)-related epitoperecognized by BR96. The high tumor specificity of BR96 and ability tointernalize (not previously described for monoclonal antibodies reactivewith the other Le^(y) determinant) suggests that the antibody recognizesa complex epitope, a portion of which includes at least a part of aLe^(y) determinant.

EXAMPLE 8 Preparation and Characterization of BR96 F(ab′)₂ Fragments

[0434] Murine BR96 (IgG₃) was purified by Protein A affinitychromatography from murine ascites fluid. Briefly, delipidated asciteswas passed over a column containing a matrix of immobilized Protein A(RepliGen Corp., Cambridge, Mass.) previously equilibrated with 1 Mpotassium phosphate, pH 8.0. Following the passage of ascites, thecolumn was washed with equilibration buffer until no further protein wasspectrophotometrically detected. The bound BR96 was then eluted from thecolumn using 0.1 M citrate buffer, pH 3.0. Immediately after elution,the eluate was neutralized with 1.0 M Tris buffer, pH 9.0, until the pHwas approximately 7.0. The monoclonal antibody was then dialyzed intoPBS and concentrated prior to storage or use.

[0435] F(ab′)₂ fragments were then generated by digesting purified BR96monoclonal antibody with pepsin according to Lamoyi, “Preparation ofF(ab′)₂ Fragments from Mouse IgG of Various Subclasses,” Meth. Enzymol.121:652-663 (1986). Residual whole antibody and Fc fragments wereadsorbed from the reaction mixture by passage over a protein A affinitycolumn. The resulting F(ab′)₂ fragment preparations were dialyzedextensively against PBS and sterile filtered.

[0436] The BR96 F(ab′)₂ fragments preparations were characterized by gelpermeation HPLC, SDS-PAGE and by ELISA on the human breast tumor line3396 (Bristol-Myers Squibb Co., Seattle, Wash.). Gel permeation HPLC wasused to assess the molecular sizes of the proteins comprising theF(ab′)₂ preparation. Reproducible chromatograms from differentpreparations indicated that 75-80% of the protein was F(ab′)₂. Noprotein was detected at the positions representing higher molecularweight material, such as whole BR96 or protein aggregates. The remaining20-25% of the protein eluted at positions corresponding to inactivatedpepsin and to other smaller non-protein A-binding digestion products.

[0437] Nonreducing and reducing SDS-PAGE was used to examine thedenatured molecular sizes and structural arrangement of the proteins inthe F(ab′)₂ preparations. A single major band at the position of F(ab′)₂(approximately 100 kdal) was typically observed, with no visiblecontaminating whole monoclonal antibody band (160 kdal). Lower molecularweight bands (i.e., less than 100 kdal) representing inactivated pepsinand small digestion products were minimal. Under reducing conditions theexpected results were obtained with the only major bands occurring as adoublet at approximately 25 kdal representing the light chain and theremaining fragmented portion of the heavy chain. No whole heavy chainband was observed.

[0438] Functional (binding) activity of the BR96 F(ab′)₂ fragments wascompared to that of whole BR96 in an ELISA with 3396 cells supplying theantigen. Binding of BR96 whole antibody or F(ab′)₂ fragments to thecells was detected with an HRP-conjugated goat anti-murine K light chainreagent as shown in FIG. 13. On a duplicate plate, binding of whole BR96was distinguished from binding of F(ab′)₂ fragments by usingHRP-conjugated protein A which binds to the whole antibody but not theF(ab′)₂ fragments (FIG. 14).

[0439] These results indicate that BR96 F(ab′)₂ (lot R0201-1663-03, lot2) contained a trace amount of whole BR96 antibody. The level ofcontaminating whole antibody can be estimated to be approximately 8trifold dilutions away from the amount of F(ab′)₂ present, or about0.01%. The other F(ab′)₂ preparation (lot R9011-1481-48, lot 1) showedno detectable level of contaminating whole BR96, indicating that anyeffect of BR96 can be explained by binding of the Fab region and not theFc region.

[0440] In summary, the BR96 F(ab′)₂ preparations appear to be completelyfree of contaminating whole BR96 IgG by HPLC and by SDS-PAGE. In onlyone instance, when a very sensitive ELISA method was used weredetectable levels of contaminating whole BR96 antibody found and thisrepresented only approximately 0.01% by weight compared to the amount ofF(ab′)₂ fragments present.

EXAMPLE 9 Preparation and Characterization of Chimeric BR96 Antibody(ChiBR96)

[0441] The murine/human chimeric BR96 antibody of the invention(“ChiBR96”) was produced using a two-step homologous recombinationprotocol as described by Fell et al., in Proc. Natl. Acad. Sci. USA86:8507-8511 (1989) and in co-pending patent application by Fell andFolger-Bruce, U.S. Ser. No. 243,873, filed Sep. 14, 1988, and Ser. No.468,035, filed Jan. 22, 1990, and assigned to the same assignee as thepresent application; the disclosures of all of these documents areincorporated in their entirety by reference herein.

[0442] Human Heavy Chain DNA Transfection

[0443] The murine hybridoma cell line BR96, ATCC No. HB10036, obtainedas described above was transfected (8×10⁶ cells) with hγ1/HC-D(deposited at Agricultural Research Service Culture Collection, Peoria,Ill., NRRL No. B 18599) (FIG. 15) by electroporation (Gene Pulser;Biorad Laboratories, Richmond, Calif.) at 250 V, 960 μFd capacitancesetting, in isotonic phosphate buffered saline (PBS) and 30 μg/ml of thepurified 6.2 kb Xbal restricted fragment of the vector hgγ1HC-D. After48 hr cells were seeded in 96-well plates at 10⁴ cells/well. Selectionfor Neo^(R) was carried out in IMDM medium (GIBCO, Grand Island, N.Y.)containing 10% (vol/vol) fetal bovine serum (FBS) and the antibioticaminoglycoside G418. (GIBCO) at 2.0 mg/ml.

[0444] Detection of Secreted Human IaG (Hu γ1) Antibody by ELISA

[0445] Culture supernatants were screened using a sandwich ELISA assay 2weeks after transfection. Goat anti-human IgG, Fc specific (CALTAG, SanFrancisco, Calif.) was used as the capture antibody and goat anti-humanIgG, Fc specific conjugated to horseradish peroxidase HRPO, (CALTAG) wasthe antibody used to detect bound human IgG. Cells from the HuIgGpositive wells were subcloned by dilution and dilution clones werescreened by ELISA to detect human IgGγ1 by the previously describedmethod. The clones containing human IgGγ1 were also screened by ELISA todetect murine IgG3 heavy chain. Goat anti-mouse IgG3 (SouthernBiotechnology Assoc., Inc., Birmingham, Ala.) was used as the captureantibody and goat anti-mouse conjugated to HRPO (Southern BiotechnologyAssoc., Inc.) was the antibody used to detect the mouse IgG3.

[0446] One of the human IgGγ1 positive murine IgG3 negative (Huγ1⁺,MuG3⁻) clones was chosen and designated ChiHBR96. This heavy chainchimeric hybridoma cell line, ChiHBR96 was characterized for antigenspecificity on MCF-7 cells and for expression levels by a quantitativeELISA for human IgG expression on MCF7 cells. The cell line ChiHBR96expressed approximately 20 μg/ml of antigen-specific human IgG antibody.

[0447] Light Chain DNA Transfection

[0448] The ChiHBR96 hybridoma (8×10⁶ cells) was transfected byelectroporation as described above but using 30 μg/ml of the human lightchain recombination vector pSV₂gpt/C_(K) (NRRL No. B 18507) containingthe human light chain K immunoglobulin sequence shown in FIG. 16,linearized with HindIII. After 48 hr cells were seeded in 96-well platesat 10⁴ cells/well. Selection for gpt was carried out in IMDM mediumcontaining 10% (vol/vol) FES, 15 μg/ml hypoxanthine, 250 μg/ml xanthineand 2.25 μg/ml mycophenolic acid (MA).

[0449] Detection of Secreted Human Kappa (Hu K) Antibody by ELISA

[0450] Culture supernatants were screened using a sandwich ELISA assayas described above, 2 weeks after transfection. Goat α-human K (CALTAG)was the capture antibody and goat anti-human K HRPO (CALTAG) was theantibody used to detect bound human K. Wells containing human K antibodywere subcloned by dilution and the clones were screened by ELISA todetect human K or murine K chain. Goat anti-mouse K (Fisher Scientific,Pittsburgh, Pa.) was used as the capture antibody and goat anti-mouse Kconjugated to HRPO (Fisher Scientific) was the antibody used to detectthe presence of the mouse K chain. One of the human K positive, murine Knegative clones (HuK⁺, MuK⁻) was chosen to analyze antigen specificityon MCF-7 cells and for expression levels by a quantitative ELISA forhuman IgG expression on MCF-7 cells. A cell line that was antigenspecific for MCF-7 cells and HuIgG⁺, MuIgG3⁻, HuK⁺, MuK⁻ was chosen anddesignated Chimeric BR96 (ChiBR96).

[0451] The original expression of the heavy and light chain antigenspecific chimeric BR96 (ChiBR96) antibody was approximately 25 μg/ml.Through four sequential rounds of cloning the line in soft agarose witha rabbit α HuIgG antibody overlay to detect cells secreting the highestamount of chimeric antibody (Coffino et al., J. Cell. Physiol.79:429-440 (1972)), a hybridoma cell line (ChiBR96) was obtainedsecreting approximately 130 μg/ml of chimeric antibody. HybridomaChiBR96 was deposited with the ATCC on May 23, 1990, and there providedwith the deposit number, ATCC No. HB 10460.

[0452] Binding of ChiBR96

[0453] The relative affinity of the ChiBR96 antibody and murine BR96antibody of the invention for the tumor associated antigen on MCF-7cells was determined by an ELISA competition binding assay (Hellstrom etal., Cancer Res. 50:2449-2454 (1990)). Briefly, adherent antigen bearingcell line MCF-7 was plated in a 96-well microtiter dish at 3×10⁴cells/well and allowed to grow to confluency for about 3-4 days. Thegrowth media was discarded and the cells are fixed with 0.5%glutaraldehyde in PBS (Sigma Chemical Co., St. Louis, Mo.), at 100μl/well for 30 min. The glutaraldehyde was discarded and the plate waswashed gently with PBS three times. The plate was then blocked withbinding buffer (0.1% BSA in DMEM) 200 μl/well for 1 hr or was storedindefinitely at −20° C. Binding buffer was discarded and samples andstandards were added to the wells. The plates were covered and incubatedovernight at 4° C. Samples and standards were discarded and the plateswere washed three times with PBS. HRP-conjugate diluted in 1% horseserum in PBS was added to wells, 100 μl/well and incubated for 1 hr at37° C. The ELISA was developed with 3,3′,5,5′-tetramethyl benzidine(TMB) chromagen (Genetic Systems, Seattle, Wash.) in a citrate buffer.Color development was arrested with 3N H₂SO₄ and the plate was read on aTitertek Microplate reader at 450 nm. This assay determined how well 0.3μg/ml of biotinylated ChiBR96 antibody competes with either unlabeledChiBR96 or unlabeled murine BR96 monoclonal antibody for the antigen.The bound biotinylated ChiBR96 antibody was detected with avidin-HRPOand developed with standard ELISA reagents.

[0454] As shown in FIG. 17, the overlap of the two binding curvesindicates that the two antibodies have the same specificity and relativeaffinity for the tumor antigen.

EXAMPLE 10 Characterization of the ChiBR96 Antibody and BR96 F(ab′)₂Fragments

[0455] Cytotoxicity of Unmodified ChiBR96 and BR96 F(ab′)₂ Fragments

[0456] Living suspended cells from the BR96 antigen positive carcinomalines 3396, 2987 and MCF-7, were treated with ChiBR96 and BR96 F(ab′)₂fragments prepared as described in Examples 8 and 9, above, to determinecytotoxicity of these antibodies as compared to the BR96 monoclonalantibody of the invention. The cytotoxicity tests were performed by FACSassay as described above in Example 4. The results of these experimentsare shown in FIGS. 18-20 as percentage dead cells vs. antibodyconcentration in μg/ml.

[0457]FIG. 18 and 20 show that the chimeric BR96 antibody and F(ab′)₂fragments of BR96 IgG3 are similar to BR96 monoclonal antibody withrespect to cytotoxicity to 3396 and MCF-7 cells. FIG. 19 demonstratesthat the cytotoxic effect on 2987 cells is much lower than on the otherbreast carcinoma cells (FIGS. 18 and 20). These results suggest that ahigher binding ratio (Table 2) is important for killing by theseantibodies and/or that different tumor cells might have differentsensitivity to killing by these antibodies. These results illustratethat the ChiBR96 antibody and the F(ab′)₂ fragments are cytotoxic bythemselves, i.e., in unconjugated form, and also illustrate that thecytotoxicity of the BR96 antibodies is not dependent on the Fc region.

[0458] Internalization of ChiBR96

[0459] The internalization of the ChiBR96 antibody within carcinomacells was evaluated in comparison to internalization of the BR96monoclonal antibody. The antibodies were conjugated to ricin A chaintoxin to form immunotoxins ChiBR96-RA (1-4 Ricin A chains per antibodymolecule) and BR96-RA (1-2 Ricin A chains per antibody molecule) andinternalization by carcinoma cell lines 3396 and 3630 was measured usinga thymidine uptake inhibition assay, as described in Example 3, above.

[0460] Graphs of the percent inhibition of thymidine incorporation vs.immunotoxin concentration for each cell line tested are shown in FIGS.21 and 22. FIG. 21 depicts the percent inhibition of thymidineincorporation by cells from the 3396 breast carcinoma cell line causedby internalization of ChiBR96-RA and BR96-RA. As shown in the graph,ChiBR96 is internalized similarly to BR96, and appears to be at least asefficient as BR96 at killing tumor cells. Similar results were obtainedwith the 3630 breast carcinoma cell line (FIG. 22).

[0461] Antibody-Dependent Cellular Cytotoxicity Activity of ChiBR96Antibody

[0462] Determination of antibody-dependent cellular cytotoxicityactivity of ChiBR96 was conducted as described in Example 5, above usingthe following cell lines: breast cancer lines 3396, 3630 and 3680(Bristol-Myers Squibb Co., Seattle, Wash.) and MCF-7 (ATCC No. HTB22);ovarian cancer line 3633-3 (Bristol-Myers Squibb Co., Seattle, Wash.);and lung cancer lines 2987; 3655-3 and 2981 (Bristol-Myers Squibb Co.,Seattle, Wash.). The results are shown in Table 3 for various antibodyconcentrations. TABLE 3 ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITYACTIVITY OF CHIBR96 Antibody Concentration (μg/ml) Cell Lines AntibodyNK 10 1 0.1 0.01 0.001 Breast Cancer 3396 BR96 28 86 74 58 27 25 ChiBR9688 79 60 34 26 MCF-7 BR96 16 82 69 54 17 15 ChiBR96 90 82 57 25 17 MCF-7BR96 22 73 69 48 22 22 ChiBR96 76 70 57 33 26 3630 BR96 30 69 64 42 3034 ChiBR96 69 56 42 36 36 3680 BR96 13 73 67 58 34 38 ChiBR96 70 71 6139 30 Ovarian Cancer 3633-3 BR96 20 92 90 64 28 23 ChiBR96 88 88 54 4329 Lung Cancer 2987 BR96 11 51 57 41 9 7 ChiBR96 69 65 51 28 15 3655-3BR96 4 49 37 0 0 0 ChiBR96 39 35 12 6 5 2981 BR96 3 4 3 3 4 5 ChiBR96 54 3 4 4

[0463] The results shown in Table 3 for various antibody concentrationsindicate that ChiBR96 mediates antibody-dependent cellular cytotoxicityactivity to a similar extent as BR96. The antibody-dependent cellularcytotoxicity activity can be seen at antibody concentrations lower thanthose at which the ChiBR96 antibody is cytotoxic by itself. Whenantibody BR96 was used alone as a control it produced 0% killing at theconcentrations tested. Antibody-dependent cellular cytotoxicity activitywas only found with the BR96 antibody-binding cell lines.

[0464] Ability of ChiBR96 to Mediate Complement-Mediated Cytotoxicity

[0465] Determination of the ability of ChiBR96 to kill tumor cells inthe presence of human serum as a source of complement(complement-dependent cytotoxicity) were performed as described inExample 6, using breast cell lines 3396; MCF-7, 3630 and 3680; ovariancancer cell line 3633-3; and lung cancer cell lines 3655-3, 2987 and2981. Table 4 presents the results. TABLE 4 COMPLEMENT-DEPENDENTCYTOTOXICITY ACTIVITY OF CHIBR96 ANTIBODY CONCENTRATION (μG/ML) CellLines Antibody 10 1 0.1 0.01 Breast Cancer 3396 BR96 100 99 78 13ChiBR96 86 92 13 2 MCF-7 BR96 94 100 63 2 ChiBR96 92 83 1 0 3630 BR96 94100 82 9 ChiBR96 86 86 33 9 3680 BR96 100 100 19 7 ChiBR96 87 100 5 9Ovarian Cancer 3633-3 BR96 98 98 21 0 ChiBR96 100 100 26 1 Lung Cancer3655-3 BR96 91 22 0 0 ChiBR96 46 3 0 0 2987 BR96 100 100 1 0 ChiBR96 10043 0 0 2981 BR96 0 3 3 2 ChiBR96 1 1 2 10

[0466] As shown in Table 4, ChiBR96 gave a cytotoxic effect(complement-dependent cytotoxicity) similar to that of BR96, in thepresence of human serum containing complement. BR96 and ChiBR96 were notcytotoxic in any concentration. Human serum was also not cytotoxic.

[0467] The above results demonstrate that the whole BR96 antibody andchimeric antibody of the invention are internalized within carcinomacells to which they bind, are cytotoxic alone in unmodified form andhave antibody-dependent cellular cytotoxicity and complement-dependentcytotoxicity activity for cells expressing a higher amount of epitopes.

EXAMPLE 11 Evaluation of BR96 Antibodies In Vivo

[0468] The therapeutic potential of the unmodified BR96 antibody of theinvention for treatment of tumors was examined in a series ofexperiments using human tumor xenografts in nude mice. In addition,certain parameters were examined that might influence the efficacy ofBR96 as an antitumor agent. These parameters include level of antigenexpression on the target tumor line, time from tumor implantation toinitiation of therapy and effects of dose.

[0469] In all the in vivo experiments of this example, the requirednumber of Balb/c nu/nu mice (Harlan Sprague Dawley, Indianapolis, Ind.)were implanted with either the human lung adenocarcinoma cell line H2987or H2707 tumor line. Cells from these tumor lines were grown in vitro,harvested, washed and resuspended in PBS prior to subcutaneous (s.c.)implantation of 10 million cells into the rear flank of each mouse.These groups of mice were then randomized and separated into smallerequal groups of 8 or 10 mice each.

[0470] To increase the chance of observing any antitumor effects of BR96while still requiring the antibody to actually localize to the tumorimplant site for any effect to occur, therapy was initiated 24 hoursafter tumor implantation on day 2. Both the BR96 and control monoclonalantibodies were administered at the same dose and schedule, althoughinitiation of therapy in some cases varied. The treatment dose wasadministered in 0.2 ml PBS intravenously (i.v.) through the tail vein ofthe mouse. Normally the schedule was once every three days for fiveinjections (Q3DX5). However, two extra injections were given on days 19and 21 after H2987 tumor implantation in the initial experiment.

[0471] Antitumor Effects of BR96 Antibody in 2987 and 2707 Tumors

[0472] Tumor volumes were determined for each animal weekly withmeasurement usually beginning on the eighth day after implantation.Tumor volumes were calculated from the measurements of tumor length andperpendicular width using the formula:

Tumor volume=longest length×(perpendicular width squared/2)

[0473] Group mean values were then calculated and plotted against timeafter tumor implantation.

[0474] In the initial experiment depicted in FIG. 23 treatment with BR96resulted in highly significant anti-tumor effects against the H2987 cellline. BR64, which also binds and is internalized by these cells, wasused as a negative control, and showed little if any effect compared tothe PBS treated controls.

[0475] Table 5 summarizes the effects on the individual tumors at theend of treatment in this first experiment. TABLE 5 EFFECTS OF TREATMENTWITH UNMODIFIED BR96 INITIATED AT DIFFERENT TIMES AFTER H2987IMPLANTATION EXPERIMENT 1 DAY 28 TUMOR RESPONSE GROUP MAb COMPLETEPARTIAL STABLE PROGRESSION 1 BR96 2 0 3 5 2 BR64 0 0 1 9 3 PBS 0 0 0 10

[0476] Only treatment with BR96 antibody resulted in complete absence oftumor. Two animals in this group were tumor free and an additional 3animals showed cessation of growth of their tumors following treatmentwith BR96 antibody. The two mice showing no signs of tumor remainedtumor free throughout the course of the experiment.

[0477] Antitumor Effects of BR96 Antibody on Established Tumors

[0478] One of the ultimate goals of tumor therapy is the effectivetreatment of established and growing tumors. To examine whether BR96could have an antitumor effect on established tumors the H2987 or H2707lung adenocarcinoma tumor lines were used as xenografts in nude mice.Because both of these tumor lines result in palpable tumors eight daysafter administration of 10 million cells s.c., delaying initiation oftreatment provided a method to examine antitumor effects on establishedtumors.

[0479] Therefore, to further examine the efficacy of unmodified BR96,several experiments were performed where treatment was withheld foreither 5 or 8 days following s.c. tumor implantation. The delay intreatment initiation allowed the tumor cells to become establishedtumors. This results in an animal model that is more difficult to treatbut resembles the clinical situation in a more realistic manner.

[0480] The treatment protocol is summarized in Table 6. Three groups of10 mice each were treated with BR96 antibody initiated at differenttimes as described in this Table. Control mice received either FA6 orPBS beginning on DAY 2. FA6 is a murine IgG₃ directed against abacterial antigen not found in mammalian species, and acted as anisotype matched nonbinding negative control monoclonal antibody. TABLE 6EFFECTS OF TREATMENT WITH UNMODIFIED BR96 INITIATED AT DIFFERENT TIMESAFTER H2987 OR H2707 IMPLANTATION TREATMENT PROTOCOL DAYS GROUP MAbSCHEDULE/ROUTE DOSE INJECTED 1 BR96 Q3DX5 i.v. 1 mg, 2, 5, 8, 11, 14 2BR96 Q3DX5 i.v. 1 mg 5, 8, 11, 14, 17 3 BR96 Q3DX5 i.v. 1 mg 8, 11, 14,17, 20 4 FA6 Q3DX5 i.v. 1 mg 2, 5, 8, 11, 14 5 PBS Q3DX5 i.v. 0.2 ml 2,5, 8, 11, 14

[0481] The results of this treatment protocol for both H2987 and H2707tumor cell lines are shown in FIG. 24, where the number of animalswithout tumors versus when initiation of treatment after tumorimplantation occurred are plotted. Absence of tumor, as defined by theabsence of a palpable tumor, was assessed at the end of treatment foreach group. The day used for the determination of tumor absence variedsince treatment was initiated at different times post tumor implant.Early initiation of treatment was clearly more effective and efficacydecrease as onset of treatment increased from time of tumor implant.Since delay in initiation of treatment allows greater growth andestablishment of the tumor, decreased efficacy at later treatmentinitiation times reflects the increasing difficulty of treating largerand more established tumors.

[0482] These results demonstrate that BR96 has antitumor effects againsttwo different tumor cell lines. Antitumor effects were only observed inthe three groups treated with BR96 antibody while those animals treatedwith either the control FA6 or PBS showed no antitumor effects.

[0483] It is significant that the differences in efficacy with moreestablished tumors are greater with the higher antigen expressing tumorline, H2707. The observation that H2707 has a greater response to BR96therapy than H2987 is consistent with the assumption that the amount ofantigen expressed by a tumor cell may influence the efficacy of BR96treatment. From the data above it is clear that BR96 has antitumoreffects against staged tumors.

[0484] Dose Effects of BR96 Antibody

[0485] In another experiment, the dose effects of BR96 against the H2707tumor line were examined. In this experiment, BR96 was administered indecreasing half log amounts from 1 mg/dose to 0.032 mg/dose. The meantumor volumes versus time post tumor implant of the groups are presentedin FIG. 25. The control treated animals were given only the highest doseof monoclonal antibody, 1 mg/dose FA6. These control animals showed noantitumor effects while there was a dose dependent response when BR96antibody was administered over the chosen dose range.

[0486] Antitumor Effects of F(ab′)₂ Fragment and Chimeric BR96

[0487] In addition, antitumor effects of the F(ab′)₂ BR96 fragment wereexamined to determine if the antitumor effects seen in vivo were due tothe Fc portion or if actual binding to the tumor with its subsequentinternalization was sufficient for cell death, as indicated by in vitroassays. The dose of F(ab′)₂ fragment was 0.66 mg/dose using the sameschedule as the whole BR96. This dose corresponds to an approximatemolar equivalent of binding regions compared to the 1.0 mg/dose wholeIgG₃ BR96. Mean tumor volume values versus time post tumor implantationfor this group treated with the antibody fragment are shown in FIG. 26.There were clearly some antitumor effects although the effects were notas strong as with whole antibody. These effects were most pronounced atthe earlier time points during and immediately following treatment.

[0488] Chimeric BR96 was also examined for antitumor effects in thisexperiment. An intermediate dose of 0.32 mg/dose for the chimericmonoclonal antibody was chosen. The mean tumor volume values for thisgroup of mice is also shown in FIG. 26. Treatment with chimeric antibodyBR96 was more efficacious than a comparable dose of the murine BR96IgG₃. This is further demonstrated in FIG. 27 which shows that 6 of the8 mice treated with chimeric BR96 were free of palpable tumors at theend of treatment.

[0489] Examination of the individual tumors depicted in FIG. 27 showsthat at completion of treatment a clear dose effect was evident by thenumber of animals without tumors after treatment with decreasing amountsof whole IgG3 BR96 antibody from 1.0 to 0.1 mg/dose. Surprisingly,treatment with 0.032 mg/dose resulted in an antitumor effect similar tothe 0.32 mg/dose. This may reflect that the level of cells killed in thetumor from the treatment was very close to the minimum amount necessaryfor the tumor to continue to grow.

[0490] Three of the eight animals treated with the F(ab′)₂ fragment werefree of palpable tumors after treatment. Therefore the Fc portion is notentirely necessary for the antibody to have antitumor effects in vivoalthough it should enhance the tumorcidal properties of BR96,particularly in immunocompetent animals.

[0491] The above results demonstrate that unmodified BR96 antibodies areeffective antitumor agents against tumor lines in vivo. Moreover, theBR96 antibodies have an effect on staged or established growing tumors.There is an indication that higher antigen density on the tumor line mayincrease the ability of BR96 to kill these cells. It has been shown thatany of the forms of the monoclonal antibody, i.e., chimeric, marinewhole IgG₃ or F(ab′)₂ fragments, are effective as antitumor agents.Earlier treatment and higher doses are preferred.

EXAMPLE 12 Localization and Biodistribution of BR96 Antibodies

[0492] Radioiodinated BR96 monoclonal antibodies administered at dosesused in the therapy experiments described above in Example 11 were usedto determine biodistribution characteristics. Specifically, the wholeIgG₃ BR96, chimeric or F(ab′)₂ fragments together with the appropriatecontrol (whole monoclonal antibody FA6, chimeric 96.5 and 96.5 F(ab′)₂,respectively) were used to localize in the tumor and various otherorgans.

[0493] Prior to the localization experiments, animals were injected withtumor cells as described above in Example 11, for the therapy studies.However, the tumors were allowed to grow in the animals forapproximately 2 weeks. At this time, 100 μg of BR96 antibody or fragmentwas radiolabeled with ¹²⁵I using 10 μg Iodogen for 10 minutes at roomtemperature as directed by the manufacturer. Control antibody orfragments were labeled with ¹³¹I using the same method. Theseradioiodinated antibodies were diluted with the appropriate unlabeledantibodies to reach the doses used in the therapy experiments. Both thespecific and nonspecific antibodies were then mixed and administeredtogether, i.v., through the tail vein of the mouse. At selected timesmice were randomly pulled from the group, anesthetized, bled through theorbital plexus and sacrificed. Various tissues were removed, weighed andcounted on a gamma counter capable of differentiating between the tworadioisotopes of iodine. From this data, percent injected dose andpercent injected dose per gram were calculated.

[0494] The accumulated data from the 24 post administration time pointin the localization experiments are summarized in Table 7. TABLE 7SUMMARY OF BIODISTRIBUTION EXPERIMENTS % INJECTED DOSE/GRAM 24 HRS. POSTADMINISTRATION DOSE TUMOR ANTIBODY (mg) CELL BLOOD TUMOR LIVER SPLEENKIDNEY LUNG 1) BR96-G₃ 1.0 H2987 10.2 6.8 2.2 1.9 3.4 4.7 FA6 1.0 6.32.1 2.1 1.6 2.4 3.2 2) BR96-G₃ 0.3 H2707 9.0 7.0 1.8 1.6 2.7 3.7 FA6 0.35.9 2.7 2.0 1.8 2.2 2.8 3) ChiBR96 0.32 H2707 7.2 8.2 1.4 1.6 2.0 3.5Chi96.5 0.32 7.5 2.3 1.8 1.6 1.9 3.5 4) F(ab')₂ BR96 0.65 H2707 <0.3<0.3 <0.3 <0.3 <0.3 <0.3 96.5 0.65 <0.3 <0.3 <0.3 <0.3 <0.3 <0.3

[0495] The only tissue showing significant differences between specificand nonspecific antibody is the tumor. All other tissues examined showapproximately equal uptake between the specific and nonspecificantibodies. One possible exception is the lower blood levels for thenonspecific antibody, FA6. This indicates accelerated blood clearance ofthis antibody. However, the difference between the specific andnonspecific antibody in the tumor is greater than the difference inblood levels between the FA6 and BR96 antibodies.

[0496] The data in Table 7 also demonstrate that the percent of the dosepresent in a particular organ is constant regardless of the doseadministered. This would therefore indicate there is quantitatively moreantibody present at the tumor site when higher doses are administered.In addition, there are no apparent differences between the two tumorlines with respect to specific vs nonspecific uptake.

[0497] Table 7 also demonstrates that the F(ab′)₂ was cleared from theanimal at a much faster rate than either the IgG₃ or chimeric BR96. Thiscould explain the reduction in efficacy of the fragment compared towhole antibody in the therapeutic experiments. Any antitumor effectsfrom the fragment must therefore be rapid and occur during the shorttime span prior to being cleared.

[0498] ChiBR96 localized at a comparable level to the IgG₃ BR96. Higheramounts were present only in the tumor compared to the control chimericantibody. This suggests that any increase in efficacy of the chimericantibody compared to the murine BR96 IgG₃ is due to the human constantregion substitution. Of equal importance, the human constant regionsubstitution does not appear to affect the ability of the chimericantibody to localize to the tumor or adversely affect itsbiodistribution.

[0499] In summary, the IgG₃ and chimeric forms of BR96 are capable ofspecifically localizing to the tumor site. Moreover, both localizationand therapeutic effects have been shown in these preliminary experimentsat comparable doses. Indirect evidence of localization of the F(ab′)₂fragments was shown by the antitumor activity of the fragments in thetherapy experiments. This activity must occur before 24 hours.

EXAMPLE 13 Preparation and Cytotoxicity of Chimeric Monoclonal AntibodyF(ab′)₂ and Fab′ Fragments Conjugated to Pseudomonas Exotoxin

[0500] Cell-specific cytotoxic reagents were prepared by chemicallycombining the chimeric antibody BR96 (ChiBR96) with Pseudomonas exotoxinA (PE) using either native PE or a truncated form (LysPE40) devoid ofthe cell recognition region (Domain I). A variety of chimericBR96-immunotoxins were constructed by chemical conjugation of PE andLysPE40 with Fab, or F(ab′)₂ enzymatic digest products, or BR96antibody, by thiolation with 2-iminothiolane or by direct attachment tointact BR96 antibody by reduction with DTT as described below.

[0501] Reagents

[0502] Succinimidyl 4-(N-maleimido-methyl) cyclohexane 1-carboxylate(SMCC) and 2-iminothiolane (2-IT) were purchased from the PierceChemical Corporation (Rockford, Ill.). Soluble pepsin was purchased fromSigma Chemical Co. (St. Louis, Mo.). Na(¹²⁵I ) and (³H)-leucine werepurchased from New England Nuclear (Boston, Mass.). Native PE waspurchased from Bema Products (Coral Gables, Fla.). Mono Q columns werepurchased from Pharmacia (Uppsala, Sweden). TSK-3000 columns werepurchased from TosoHaas, Inc. (Philadelphia, Pa.). Immunoblots wereperformed using mouse (anti-id BR96) and rabbit (anti-PE) ABC kits(Vector Laboratories, Burlingame, Calif.). Rabbit polyclonal anti-PEantibody and mouse anti-PE monoclonal antibody M40/1 were supplied byDrs. Ira Pastan and David FitzGerald, National Institutes of Health(Bethesda, Md.). Anti-idiotypic BR96 antibody 757-4-1 was prepared usingthe BR96 antibody of the invention and standard procedures for preparinganti-id antibodies (see, Kahn et al., Cancer Res. 49:3159-3162 (1989)and Hellstrom et al., Cancer Res. 50:2449-2454 (1990)) by Dr. BruceMixan, Bristol-Myers Squibb (Seattle, Wash.)).

[0503] Cell Culture and Plasmids

[0504] All cells were cultured in RPMI 1640 supplemented with 10% fetalbovine serum, except L929, which was cultured in DMEM supplemented with10% fetal bovine serum. Plasmid pMS8 (FIG. 36), which encodes the genefor LysPE40 under control of the T7 promoter, was constructed by Dr.Clay Siegall (provided by Dr. Ira Pastan, NIH, Bethesda, Md.) from thevector pVC85 (Kondo et al., J. Biol. Chem. 263:7470-7475 (1988)) byinserting at the amino terminus a lysine residue and also inserting amultiple cloning site.

[0505] Expression and Purification of LysPE40

[0506] The plasmid pMS8 encoding LysPE40 was transformed into E. coliBL21 λDE3) cells and cells were cultured in Super Broth (Digene, Inc.,Silver Spring, Md.) containing 75 μg of ampicillin per ml at 37° C. Whenabsorbance at 650 was 2.0 or greater, isopropyl1-thio-β-D-galactopyranoside was added (1 mM) and cells were harvested90 minutes later. The bacteria were washed in sucrose buffer (20%sucrose, 30 mM Tris-HCl (pH 7.4), 1 mM EDTA), and osmotically shocked inice-cold H₂O to isolate the periplasm. LysPE40 protein was purified fromthe periplasm by successive anion-exchange and gel-filtrationchromatographies using a Pharmacia fast protein liquid chromatography(FPLC) system as described previously (Batra et al., Proc. Natl. Acad.Sci. USA 86:8545-8549 (1989) and Siegall et al., Proc. Natl. Acad. Sci.USA 85:9738-9742 (1988)).

[0507] Generation of BR96 F(ab′)₂ and Fab′ Fragments

[0508] F(ab′)₂ fragments were generated from ChiBR96 (4 mg/ml) by pepsindigestion (25 μg/ml in 0.1 M citrate buffer, pH 4.0, (Parham, inHandbook of Experimental Immunology, Weir, Ed., Blackwell ScientificPublishers, p. 1-23 (1986)). After 6 hours incubation at 37° C.,digestion was terminated by adjusting the pH to 7.2 with PBS. Purity ofthe digest preparation was 90-95% F(ab′)₂ determined by SDS-PAGE (4-20%gradient gels) and Coomassie blue staining.

[0509] Fab′ was prepared from the ChiBR96 F(ab′)₂ by reduction withcysteine to break the remaining interchain disulfide bonds (Parham etal., supra). Briefly, F(ab′)₂ molecules (2-4 mg/ml) in 0.1 M Tris-HCl(pH 7.5) were incubated at 37° C. for 2 hours with cysteine (0.01 Mfinal concentration). Free sulfhydryl groups on the Fab′ molecule werealkylated with 0.02 M iodoacetamide (CalBiochem, San Diego, Calif.) for30 minutes at room temperature to prevent recombination of the Fab′ toF(ab′)₂. The reaction mixture was dialyzed against PBS. Purity wasgreater than 85% as assessed by SDS-PAGE.

[0510] Immunotoxin Construction and Purification

[0511] Chimeric BR96 (6-10 mg/ml) was thiolated by addition of a 3-foldmolar excess of 2-iminothiolane (2-IT) in 0.2 M sodium phosphate (pH8.0), 1 mM EDTA for 1 hour at 37° C. (Batra et al., Proc. Natl. Acad.Sci. USA 86:8545-8549 (1989)), which introduces sulfhydryl groups byreaction of 2-iminothiolate with primary amines. Unreacted 2-IT wasremoved by PD-10 column chromatography (Pharmacia). Alternatively, freethiol groups were generated by reduction with dithiothreitol (DTT).Chimeric BR96 was incubated with a 20-fold molar excess of DTT for 2.5hours at 42° C. Excess DTT was removed by overnight dialysis against PBSunder nitrogen. The number of thiol groups on the monoclonal antibodywas determined by DTNB reduction (Ellman's reagent, Sigma Chem. Co.) asdescribed by Deakin et al., Biochem. J. 89:296-304 (1963), incorporatedby reference herein. This procedure routinely gave 4 thiol groups perBR96 antibody, with no reduction in antibody binding reactivity orprotein concentration. The procedure was not used with F(ab′)₂ or Fab′fragments.

[0512] Thiolated BR96 antibody was condensed with maleimide-modified PEor LysPE40. Anon-cleavable maleimide group was attached to lysineresidues on the toxin (PE or LysPE40; 6-8 mg/ml) by mixing with 3-foldmolar excess of SMCC in 0.2 M sodium phosphate (pH 7.0), 1 mM EDTA atroom temperature for 30 minutes and purified on a PD-10 column. Modifiedtoxin and thiolated antibody were mixed in a 4:1 molar ratio andincubated at room temperature for 14-16 hours to allow a thioetherlinkage to form. Immunotoxins were purified by anion-exchange (Mono Q)to remove unreacted antibody and gel-filtration chromatography(TSK-3000) to remove unconjugated toxin as previously described by Kondoet al., J. Biol. Chem. 263:9470-9475 (1988); and Batra et al., Proc.Natl. Acad. Sci. USA 86:8545-8549 (1989), all incorporated by referenceherein.

[0513] Chimeric BR96 IgG-LysPE40 (190 kDa), Fab′-LysPE40 (96 kDa) andF(ab′)₂-LysPE40 (145 kDa) conjugates were additionally analyzed bynon-reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) todetermine the size of the native conjugate (FIG. 28). From the Coomassieblue stained gels, it was determined that there was less than 5%unconjugated antibody after purification.

[0514] Binding Studies

[0515] For competition binding studies, L2987 cells (Bristol-MyersSquibb Co., Seattle, Wash.) were removed from monolayer culture using0.2% trypsin and washed with RPMI 1640 containing 2% FCS (wash buffer).Cell suspensions (1.0×10⁶ cells/0.1 ml) were incubated with 0.1 mlfluorescein isothiocyanate (FITC)-labeled ChiBR96 (13.3 μg/ml finalconcentration) and 0.1 ml of diluted antibody or immunotoxin at 4° C.for 1 hour, washed, and the amount of cell-bound FITC labeled-ChiBR96was quantified on an EPICS V model 753 Flow Cytometer (Coulter Corp.,Hialeah, Fla.).

[0516] For direct binding studies, two-fold serially dilutedimmunotoxins or antibody was incubated for 1 hour at 4° C. in 0.2 mlwash buffer containing 1×10⁶ L2987 cells. Cells were washed and thenincubated in wash buffer containing 1:40 diluted FITC labelled goatanti-human kappa antibody (Bethyl Labs, Montgomery, Tex.) for anadditional 30 min at 4° C. to quantitate cell-bound antibody. Cells werewashed and analyzed for cell surface fluorescence on a flow cytometer todetermine the amount of immunotoxin or antibody remaining on the cellsurface.

[0517] Two methods were used to determine whether there was analteration in antibody binding activity after conjugation to PE orLysPE40. Competition binding analysis showed that both immunotoxinscompeted with FITC-labeled ChiBR96 as efficiently as unconjugatedChiBR96 antibody (FIG. 29), indicating that binding affinity for theBR96 antigen was not perturbed after chemical conjugation. Similarresults were obtained using the direct binding assay for both PE andLysPE40 conjugates.

[0518] Binding activities of LysPE40 conjugated and unconjugated IgG,F(ab′)₂ and Fab′ were also compared by direct binding to L2987 tumorcells. Cell-bound antibody protein was quantitated using FITC-labeledgoat anti-human kappa light chain antibody. Binding of the LysPE40immunotoxin was similar to that obtained using unconjugated ChiBR96antibody (FIG. 30A) and agreed with results obtained using thecompetition binding assay (FIG. 29). FIG. 30B compares the bindingactivity of intact IgG to F(ab′)₂ and F(ab′)₂-LysPE40. There was no lossin immunoreactivity with the F(ab′)₂ and F(ab′)₂-immunotoxin as comparedto ChiBR96 IgG. Conjugation of PE40 to Fab′ did not affectimmunoreactivity (FIG. 30C), however, binding of the Fab′ wassignificantly decreased as compared to intact IgG (FIG. 30C), mostlikely because of the monovalency of the Fab′ molecule.

[0519] Antigenic Modulation and Internalization

[0520] Modulation of intact ChiBR96, F(ab′)₂ or Fab′ immunotoxins wasassayed on L2987 cells propagated as 90-95% confluent monolayer culturesin 96 well microtiter plates as described above. Target cells werepulsed for 1 hour at 4° C. with 0.1 ml of two-fold serially dilutedimmunotoxin ranging from 5-800×10⁷ M antibody protein in binding buffer.Monolayer cultures were washed free from unbound material using growthmedium and individual plates were incubated in complete medium undereither non-modulating (4° C.) or modulating (37° C.) conditions.

[0521] The amount of membrane-associated immunotoxin bound to targetcell populations at each time point shown in FIG. 31 was quantifiedusing (¹²⁵I)M40/1 (anti-PE) antibody (provided by Dr. D. Fitzgerald,NCI, Bethesda, Md., (Ogata et al., Inf. and Immun. 59:407-414 (1991))).Epitope mapping of M40/1 antibody determined that it binds to a 44 aminoacid region in the PE domain II (Ogata et al., supra). Monoclonalantibody M40/1 was radioiodinated using Na[²⁵I] (New England Nuclear,Boston, Mass.) and chloramine T (Kodak Chemical Co., Rochester, N.Y.) asdescribed by McConahey and Dixon, Arch. Allergy Appl. 29:185-188 (1966),incorporated by reference herein. Radioiodinated M40/1 was separatedfrom unbound iodine by PD10 column chromatography (Pharmacia). Specificactivities ranged from 2 to 5×10⁵ CPM/μg.

[0522] At various times during incubation at 37° C. or 4° C., atriplicate set of wells were twice washed with wash buffer andpulse-labeled with 0.1 ml (¹²⁵I)-labeled M40/1 antibody (0.5 μg/ml inwash buffer containing 0.2% sodium azide) to determine membrane boundconjugate. After 15 minutes, monolayers were washed free of unboundlabel, and cell-bound cpm was determined by solubilization of the cellmonolayer with 0.5 N NaOH. Cell-bound radioactivity was determined usinga LKB model 1272 gamma counter. Non-specific binding was determined byincubation of target cells with a similar concentration of unconjugatedChiBR96. In certain experiments, unconjugated PE was used to determinebackground binding levels. (¹²⁵I)-labeled M40/1 antibody did not reactwith membrane bound antibody or PE.

[0523] The ability of ChiBR96-PE and ChiBR96-LysPE40 to induce antigenicmodulation was initially measured by determining the loss of immunotoxinfrom the cell surface membrane (FIG. 31). There was no difference inmodulation kinetics between PE or LysPE40 immunotoxins withapproximately 50% of the original cell-bound immunotoxin modulated fromthe surface membrane 30 minutes after warming to 37° C. Cells incubatedunder conditions which do not allow antigenic modulation (4° C.), showedessentially no loss of cell surface toxin within 6 hours (FIG. 31A).

[0524] In order to confirm that the loss of cell-surface immunotoxin wasdue to endocytosis, cells were incubated with a [¹²⁵I]-labeledimmunotoxin complex for 1 hour at 4° C. to permit binding, washed andwere subsequently modulated at 37° C. As shown in FIG. 31B, essentiallyall the radiolabeled immunotoxin remained cell-associated, despite theconcomitant loss from the cell-surface membrane (FIG. 31A). Thesefindings confirm that most if not all of the membrane-associated BR96immunotoxins were rapidly internalized, and that internalization rateswere similar for PE and LysPE40 immunotoxins.

[0525] The capacity of ChiBR96 F(ab′)₂-LysPE40 and Fab′-LysPE40immunotoxins to internalize was also determined by measuring the loss ofcell-surface immunotoxin using radiolabeled anti-PE antibody.Essentially all of the ChiBR96 immunotoxins were completely internalizedafter 4.5 hours including the Fab′-immunotoxin (Table 8). However, ratedifferences were observed. At 2.5 hours, when 76% of the intact IgGtoxin and 72% of the F(ab′)₂ were internalized, only 12% of the Fab′immunotoxin was internalized. Therefore, both IgG, F(ab′)₂ andFab′-LysPE40 immunotoxins were modulated from the cell surface membrane,but at different rates. TABLE 8 INTERNALIZATION OF BR96-IMMUNOTOXINSFROM THE CELL SURFACE MEMBRANE OF L2987 CELLS % INTERNALIZATION 2.5 hr4.5 hr BR96-LysPE40 74.0 85.0 F(ab')₂-LysPE40 72.0 91.6 Fab'-PE40 12.089.6

[0526] Inhibition of Protein Synthesis Assay

[0527] Cytotoxicity of various forms of ChiBR96 antibody conjugated toLysPE40 against tumor cells was determined by measuring inhibition ofprotein synthesis as follows: Tumor cells (1×10⁵ cells/ml) in growthmedia were added to 96 well flat bottom tissue culture plates (0.1ml/well) and incubated at 37° C. for 16 hours. Dilutions of toxin ortoxin-conjugates were made in growth media and 0.1 ml added to each well(3 wells/dilution) for 1 hour or 20 hours at 37° C. After theappropriate incubation time, unreacted material was removed by washingthe monolayer with growth media. Cells were incubated in 0.2 ml growthmedia for a total of 20 hours and pulse-labeled with [³H]-leucine (1μCi/well) for an additional 4 hours at 37° C. The cells were lysed byfreezing, thawing at 37° C. and harvested using a Tomtec cell harvester(Orange, Conn.). Cellular protein labeled with [³H]-leucine wasdetermined by counting the radioisotope using a LKB Beta Plate (LKB,Piscataway, N.J.) liquid scintillation counter.

[0528] Analysis of Competition of Immunotoxin Cytotoxic Activity

[0529] Chimeric BR96-PE40 was added at 0.8, 4 and 20 pM concentrationsto MCF-7 cells in the presence or absence of 50 μg (333 pM) ChiBR96antibody. Cytotoxicity was determined as described in the inhibition ofprotein synthesis assay as described above.

[0530] In vitro Cytotoxicity of Intact IgG-PE Immunotoxins

[0531] The in vitro cytotoxic activity of the immunotoxins againstcancer cells was assayed by comparing inhibition of protein synthesis onantigen positive and antigen negative cells (Table 9). BR96antigen-positive cell lines MCF-7, L2987, and RCA were the mostsensitive to ChiBR96-PE with EC₅₀ values of 0.14, 0.28, and 1.4 pM,respectively. The immunotoxin was also more inhibitory than native PEwhich had EC₅₀ values of 200, 140 and 380 pM. When tested onantigen-negative cell lines, little difference in EC₅₀ values between PEand the immunotoxin was observed. Specificity, (antibody-directedcell-killing), must take into account the different sensitivities of thevarious cell lines to native PE. Thus, the ChiBR96 immunotoxins were100-500 fold more potent than native PE against antigen-positive celllines. TABLE 9 CYTOTOXICITY OF CHIBR96-PE ON HUMAN TUMOR CELLS EC₅₀, pMCell BR96 DTT Reduced 2-IT-Treated Line Type Antigen ChiBR96-PEChiBR96-PE Native PE MCF-7¹ Breast Ca. + 0.10 0.14 200.0 L2987 LungCa. + 0.25 0.28 140.0 RCA Colon Ca. + 1.2 1.4 380.0 A2780 Ovarian − 23.023.0 60.0 Ca. L929 Mouse − 13.5 14.0 3.0 Fblst KB² Epider- − 220.0 231.0227.0 moid

[0532] EC₅₀ represents the amount of immunotoxin or toxin required toinhibit 50% of the protein synthesis as determined by [³H]-leucineincorporation in cellular protein. (BR96 antigen+=Epitope densityof>1×10⁴ molecules/cell).

[0533] Cytotoxicity of ChiBR96 Mab and Enzymatic Fragments Linked toLvsPE40 Against MCF-7 Cells

[0534] Smaller immunotoxin molecules may be beneficial in tumorpenetration, therefore, the cytotoxic activity of ChiBR96 as Fab′,F(ab′)₂ fragments and as an IgG linked to LysPE40 was compared (Table10). As with the ChiBR96-PE immunotoxin (Table 9), MCF-7 and L2987 cellswere the most sensitive cell lines tested. The IgG and F(ab′)₂-LysPE40molecules showed similar cytotoxic activity against MCF-7 cells(EC₅₀=8-14 pM) while the Fab′-LysPE40 conjugate was much less active(EC₅₀=780 pM) (FIG. 32). Specificity of protein synthesis inhibitionactivity of Fab′ and F(ab′)₂ conjugates was also preserved, with littleor no inhibitory activity observed against the antigen-negative celllines A2780. TABLE 10 CYTOTOXICITY OF 2-IMINOTHIOLANE SUBSTITUTEDCHIMERIC BR96-PE40 ON HUMAN TUMOR CELLS EC₅₀ pM BR96 BR96 F(ab')₂- Fab'-Cell Line Type Antigen PE40 PE40 PE40 PE40 MCF-7 Breast Ca. ++ 8 14 78015,000 L2987 Lung Ca. + 37 70 2700 17,500 RCA Colon Ca. + 84 110 500015,000 A2780 Ovarian Ca. − 650 2500 11,000 15,000 KB Epider- − >5000N.D. N.D. >25,000 moid Ca

[0535] Specificity of Growth Inhibition By ChiBR96-LysPE40

[0536] Specificity was confirmed by abrogating the protein synthesisinhibition by ChiBR96-LysPE40 with unconjugated ChiBR96. Addition ofexcess ChiBR96 antibody (50 μg) with ChiBR96-LysPE40 immunotoxin,resulted in a decrease of in vitro potency (FIG. 33). At 20 pM ofChiBR96-LysPE40, approximately 50% of its cytotoxic effect was blockedby the addition of excess unconjugated antibody, while at 4 pM, theexcess ChiBR96 completely blocked the cytotoxic activity ofChiBR96-LysPE40.

[0537] Kinetics of Cytotoxicity of ChiBR96 Immunotoxins and Native PE

[0538] In part, the effectiveness of immunatoxins may depend on the rateof internalization after binding to antigen-positive cells. To determinethe cytotoxic activity of ChiBR96-PE, ChiBR96-LysPE40 and PE, a timecourse analysis was performed where cells were incubated with toxin forup to 20 hours as described above.

[0539] After 1 hour incubation, MCF-7 cells were sensitive to ChiBR96-PEand ChiBR96-LysPE40 (EC₅₀ values of 1 and 60 pm, respectively) but notto the native toxin (EC₅₀>10,000 pM). After 20 hours MCF-7 cells wereslightly more sensitive to ChiBR96-PE and ChiBR96-PE40, but much moresensitive to PE; EC₅₀=200 pM (FIG. 34). This assay was repeated at 2, 4,and 6 hour time points. At each time point, PE was considerably lesscytotoxic against MCF-7 cells than ChiBR96-immunotoxins. This may be duein part to the mechanism by which the toxin molecule is delivered totarget cells.

[0540] This example demonstrates the production of immunotoxinscontaining the carcinoma-associated monoclonal antibody ChiBR96 andPseudomonas exotoxin A. The antibody was used in forms including nativeIgG, reduced IgG, F(ab′)₂ and Fab′. The toxin component of theimmunotoxin was either native PE or LysPE40, a truncated form containinga genetically modified amino terminus that includes a lysine residue forconjugation purposes. Chimeric BR96-toxin conjugates were found to becytotoxic to cells which display Lewis Y, a determinant recognized bythe BR96 monoclonal antibody of the invention. The most cytotoxic of theconjugates produced was ChiBR96-PE which was 1000-fold more potent thanPE itself against MCF-7 breast carcinoma cells. Chimeric BR96-LysPE40was also extremely cytotoxic towards BR96 antigen positive cells(1000-fold more potent than LysPE40). Both ChiBR96-PE andChiBR96-LysPE40 were produced using two procedures which generatedsulfhydryl groups on the antibody, by mild reduction of the antibody orby derivatizing the antibody with 2-iminothiolane. The former procedureproduced a greater yield of conjugate, but conjugates produced by bothprocedures resulted in chimeric molecules of identical activities.

[0541] Chimeric BR96-PE and ChiBR96-LysPE40 were almost fully activewith 1 hour incubation, while PE was relatively inactive (FIG. 34). Withcontinued incubation, ChiBR96-immunotoxins increase cytotoxic activityonly slightly while PE becomes cytotoxic to the MCF-7 cells at latertime points. This rapid efficacy of ChiBR96-immunotoxins is evidence ofthe utility of ChiBR96 in targeting cell populations for killing.

[0542] The binding and internalization activities ofChiBR96-immunotoxins were also examined. Immunoconjugates prepared withintact IgG or its F(ab′)₂ or Fab′ enzymatic digest products were notaffected in terms of binding by chemical conjugation to LysPE40 (FIG.30) or PE. Differences in binding activity between Fab′ and F(ab′)₂ orIgG conjugates may be attributed to differences in avidity due to themonovalence of the Fab′ molecule. We also cannot exclude the possibilitythat enzymatic digestion used to generate the Fab′ fragments contributedto the decreased avidity. Of most interest was the comparison betweenChiBR96-LysPE40 and the enzymatic fragment immunotoxins ChiBR96F(ab′)₂-LysPE40 and ChiBR96 Fab′-LysPE40. This finding is also reflectedin the cytotoxicity data (Table 10).

[0543] The results presented in this example demonstrate that bothintact PE and LysPE40 immunotoxins as well as F(ab′)₂ and Fab′immunotoxins demonstrate cytotoxic activity in vitro.

EXAMPLE 14 Preparation of Single-Chain BR96 sFv-PE40 Immunotoxin

[0544] This example describes the preparation and characterization ofcytotoxicity of a single-chain immunotoxin, BR96 sFV-PE40, consisting ofthe cloned heavy and light chain Fv portions of the BR96 monoclonalantibody of the invention linked to PE40.

[0545] Q Sepharose was purchased from Pharmacia (Uppsala, Sweden).TSK-3000 columns were purchased from TosoHaas, Inc. (Philadelphia, Pa.).Immunoblots were performed using mouse anti-idiotypic BR96 antibody757-4-1 as described above in Example 13, and ABC immunoblot kits(Vector Laboratories, Burlingame, Calif.). Chloramine T was purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). MCF-7 human breast carcinomacells were originally obtained from the ATCC (Rockville, Md.) and havebeen maintained by Bristol-Myers Squibb Company, Seattle, Wash. RCAcolon carcinoma cells were obtained from M. Brattain, Baylor University,Tex. L2987 lung adenocarcinoma cells were obtained from Dr. I.Hellstrom, Bristol-Myers Squibb Co., Seattle, Wash. A2780 ovariancarcinoma cells were obtained from K. Scanlon, NIH, Bethesda, Md., andKB epidermoid carcinoma cells were obtained from Dr. Ira Pastan, NIH,Bethesda, Md. Cells were cultured in RPMI 1640 supplemented with 10%fetal bovine serum.

[0546] Construction of BR96 sFv-PE40

[0547] In order to produce a single-chain recombinant immunotoxin, theFv domains of the light and heavy chains of BR96 IgG were isolated fromplasmid pBR96 Fv (FIG. 36) containing the BR96 Fv sequences using PCRamplification.

[0548] Identification of Primers for PCR Amplification

[0549] Two sets of PCR primers:

[0550] Primer 1: 5′-GCTAGACATATGGAGGTGCAGCTGGTGGAGTCT-3′ (SEQ ID NO: 1)and primer 2: 5′-GCTGTGGAGACTGGCCTGGTTTCTGCAGGTACC-3′ (SEQ ID NO:2) weredevised for the amplification of the V_(L) and V_(H) of murine chimericBR96 monoclonal antibody. The V_(L) and V_(H)-5′ PCR primers were basedon the N-terminal amino acid sequence of the BR96 light and heavy chains(FIG. 35, SEQ ID NO:3), respectively, while the 3′ primers were designedaccording to the frequency of the most common nucleotide at eachposition of joining (J)-region segments after alignment of V_(H) andV_(k) genes (Kabat et al., in Sequences of Proteins Of ImmunologicalInterest, U.S. Dept. Health and Human Services, Washington, D.C.(1987)). The V_(L)-5′ primer was comprised of 24 nucleotides encodingthe N-terminal amino acids of the variable light chain and a Hind IIIsite 5′ to these nucleotides while the V_(L)-3′ primer consisted of 22nucleotides which were complementary to the J region of mouse kappalight chain mRNA and a Sph I site 5′ to these nucleotides. The V_(H)-5′primer contained 30 nucleotides encoding the N-terminal amino acids ofthe heavy chain and an Eco RI site 5′ to these nucleotides while theV_(H)-3′ primer contained 22 nucleotides with J region complimentarityand a BamHI site 5′ to these nucleotides. In designing each primer,additional nucleotides were incorporated at the 5′ end in order tooptimize restriction site digestion and subsequent cloning of the PCRreaction products. The 5′ PCR primer (primer 1) was designed to encode aunique Nde I restriction site.

[0551] RNA Isolation, cDNA Synthesis and Amplification

[0552] RNA was prepared from about 1×10⁸ BR96 hybridoma cells grown inIMDM supplemented with 10% fetal calf serum (FCS). Total RNA was usedfor first strand cDNA synthesis using random hexamers at 23° C. for 10minutes in a 20 μl reaction mixture containing 1 μg of total RNA, 4 MMMgCl₂, 1 mM of each dNTP, 1 unit of RNAase inhibitor (recombinant RNAaseinhibitor originally isolated from human placenta, Perkin-Elmer/Cetus,Norwalk, Conn.), 1×PCR buffer (10×PCR Reaction buffer=500 mM KCl, 100 mMTris-HCl, pH 8.3), 2.5 μM random hexanucleotide (Perkin-Elmer/Cetus) and2.5 units of reverse transcriptase (cloned Moloney murine leukemia virus(M-MLV) reverse transcriptase, 2.5 units/μl from Perkin=Elmer/fetus).Subsequent to hexameric primer extension with reverse transcriptase, thereaction mixtures were incubated successively in a thermal cycler(Eppendorf MicroCycler) at 42° C. for 15 minutes, 99° C. for 5 minutesand 5° C. for 5 minutes.

[0553] Amplification of V_(L) and V_(H) cDNAs was performed with 35cycles of PCR using reagents according to manufacturer's instructions(GeneAmp RNA-PCR, Perkin-Elmer/Cetus) in two separate tubes with 0.15 μMeach of either V_(L)-5′ and V_(L)-3 or V_(H)-5′ and V_(H)-3′ primers.Each PCR cycle consisted of denaturation at 95° C. for 1 minute followedby annealing and extension at 60° C. for 1 minute. In order to fullyextend all cDNAs, a single held extension was performed at 60° C. for 7minutes.

[0554] Cloning of Amplified cDNA

[0555] The amplified PCR products were purified on ion-exchangemini-columns (Elutip-D, Schleicher & Schuell, Keene, N.H.), concentratedby ethanol precipitation and digested with either EcoRI and BamHI (V_(H)gene) or Hind III and Sph I (V_(L) gene). Subsequently, the digested PCRreaction products were further purified on 1.5% agarose gels (SeaKem,FMC Corp. Rockland, Me.) and the V_(H) or V_(L) gene fragmentsseparately cloned into pEV3-SM2 (Crowl et al., Gene 38:31-38 (1985))digested with either EcoRI and BamHI or with Hind III and Sph Irespectively. Clones containing V gene inserts were identified by colonyhybridization using either random-primed radiolabeled V_(L) or V_(H)cDNA fragments as probes. The nucleotide sequence was then determinedfor several cloned V_(L) or V_(H) cDNA inserts, employing primers basedupstream within the lambda P_(L) promoter or downstream of Sal I withinpBR322 sequences.

[0556] Construction of Plasmid pBW 7.0

[0557] Starting with the BR96 sFv sequence encoded by plasmid pBR96Fv(FIG. 35, prepared by Dr. McAndrew, Bristol-Myers Squibb Co.) a 550 bpsequence corresponding to the variable heavy and variable light chainsconnected with a synthetic (Gly4Ser)₃ hinge region up to the Kpn Irestriction site in the light chain, was used to PCR-amplify with primer1 and primer 2 described above. After PCR-amplification and digestionwith Nde I and Kpn I the 550 bp Nde I-Kpn I fragment was ligated into a4220 bp Nde I-Kpn I vector fragment prepared from plasmid pMS8 describedabove, (supplied by Dr. Ira Pastan, NIH, Bethesda, Md.), which encodesthe gene for PE40 under the transcriptional control of the T7 promoter(Studier et al., J. Mol. Biol. 189:113-130 (1986)). The product of thisligation was an intermediate vector designated pBW 7.01 (FIG. 35).Subsequently, the 227 bp Kpn I fragment from pBR96 Fv was subcloned intothe unique Kpn I site of pBW 7.01. The resulting plasmid pBW 7.0 (FIG.36), encoding the BR96 sFv-PE40 gene fusion, was confirmed by DNAsequence analysis.

[0558] Expression and Purification of BR96 sFv-PE40

[0559] The plasmid pBW 7.0 encoding BR96 sFv-PE40 obtained as describedabove was transformed into E. coli BL21 (λ DE3) cells cultured in SuperBroth (Digene, Inc., Silver Springs, Md.) containing 75 μg of ampicillinper ml at 37° C. When absorbance at 650 nm reached 1.0, isopropyl1-thiol-B-D-galactopyranoside (IPTG) was added to a final concentrationof 1 mM, and cells were harvested 90 minutes later. Upon induction withIPTG, the E. coli cells transformed with pBW 7.0 expressed large amountsof fusion protein that was localized to the inclusion bodies. Thebacteria were washed in sucrose buffer (20% sucrose, 30 mM Tris-HCl (pH7.4), 1 mm EDTA) and were osmotically shocked in ice-cold H₂O to isolatethe periplasm. Subsequently, inclusion bodies were isolated away fromthe spheroplast membrane proteins by extensive treatment with Tergitol(Sigma) to remove excess bacterial proteins, followed by denaturation in7 M guanidine-HCl (pH 7.4), refolding in PBS supplemented with 0.4 ML-Arginine and extensive dialysis against 0.02 M Tris, pH 7.4. Proteinwas purified using anion-exchange on a Q-Sepharose column and fractionscontaining BR96 sFv-PE40 were then pooled and separated bygel-filtration (on a TSK-3000 column) chromatographies with a Pharmaciafast protein liquid chromatograph (FLPC) system as described by Siegallet al., Proc. Natl. Acad. Sci. USA 85:9738-9742 (1988).

[0560] The chromatographic profile of the size exclusion columnindicated the presence of two major species (FIG. 37A). The firstspecies eluted between gel filtration standards of 660 kD and 158 kD andrepresents an aggregated form of the recombinant protein (fractions9-14). The second species (fractions 15-21) represents the 67 kDamonomeric form of BR96 sFv-PE40 which, as expected, eluted between the158 kDa and 44 kDa standards. These results were confirmed by reducing(FIG. 37B) and non-reducing SDS-PAGE analysis (FIG. 37C). Whereas FIG.37B shows the purification profile based on Coomassie staining, FIG. 37Cshows immunoblot analysis using anti-idiotypic BR96 antibody. The abovedata demonstrate that purification yielded two forms of recombinantprotein, monomers and aggregates.

[0561] Direct Lewis Y Determinant Binding ELISA

[0562] Because BR96 sFv-PE40 is monovalent it provides only oneantigen-binding site per molecule. In order to test the relative bindingactivities of monovalent BR96 sFv-PE40 compared to the bivalent BR96antibody, a direct binding assay was performed in which purified Lewis Ywas coated on ELISA plates and the recombinant BR96 sFv-PE40 moleculewas compared, in its ability to bind to the antigen, with severalantibodies and antibody fragments. Lewis-Y (ChemBiomed, Alberta, Canada)was diluted to 0.2 μg/ml in Coating Buffer (100 mM sodiumcarbonate/bicarbonate, pH 9.4) prior to coating Dynatech Immunon IIplates and incubating for 16 hours at 4° C. Excess antigen was removedand the plates were blocked with PTB buffer (PBS containing 0.05% Tween20 and 1% BSA) for 1 hour at room temperature followed by 3 washes withPTB. The antibody samples were serially diluted in PTB to a finalconcentration ranging from 1.25 μg/ml to 80 μg/ml and incubatedovernight at 4° C. on the plate in a volume of 50 μl/well. The plateswere washed 3 times with PTB buffer and each well was incubated with 100μl/well of biotinylated BR96 anti-idiotypic antibodies (2.56 μg/ml) inPTB for 1 hour at room temperature. The plates were then washed 4additional times with PTB. Alkaline phosphatase-conjugated streptavidin(Kirkegaard & Perry Labs, Gaithersburg, Md.) was added to each well (100μl of 0.5 μg/ml in PBS containing 1% BSA) and incubated for 1 hour at37° C. Plates were washed 3 times with PTB and 3 times with phosphatasebuffer (75 M Tris, 0.1 M NaCl, 5 mM MgCl₂, pH 9.4) and reacted withp-nitrophenyl phosphate (1 mM in phosphatase buffer) for 30 to 60minutes at 37° C. The reaction was stopped by the addition of 2 N NaOH.The plates were read at 405 nm on a Molecular Device, Inc. (Menlo Park,Calif.) microplate Reader.

[0563] In comparison with BR96 IgG, monomeric BR96 sFv-PE40 boundapproximately 5-fold less well (FIG. 38). In contrast, the aggregatedform of BR96 sFv-PE40 was unable to bind to the Lewis-Y determinant. L6IgG, an antibody that does not bind the BR96 antigen, was used as anegative control.

[0564] In addition, the competitive binding ability of BR96 sFv-PE40 wascompared with BR96 IgG. Microtiter plates were coated with Lewis-Yantigen as described above. Antibody samples were diluted in PBScontaining 1% BSA to final concentrations ranging from 1.36 μg/ml to 175μg/ml. ¹²⁵I-BR96 IgG was added to each sample (5 μCi/ml) along withantibody competitor to a final volume of 100 μl. The entire mixture ofradiolabeled BR96 IgG and antibody competitor were added to the Lewis-Ycoated plates and incubated for 2 hours at 37° C. The plates were washedfive times with PBS containing 0.05% Tween-20 and the wells counted on agamma counter. This assay, which also used Lewis-Y coated plates,measured the amount of bound radioiodinated BR96 IgG when compared withvarious amounts of BR96 sFv-PE40 or BR96 IgG.

[0565] The results of the competitive binding assay were that BR96sFv-PE40 competed 5-fold less well than BR96 IgG (FIG. 39) whichcorrelates with the direct binding data in FIG. 38. The addition of L6IgG, which did not compete for binding, demonstrates the specificity ofthis assay.

[0566] Cytotoxicity of BR96 sFv-PE40 against Cancer Cells

[0567] To determine the cytotoxic potential of monomeric BR96 sFv-PE40the effect of the single-chain immunotoxin was compared to that of thechemical conjugate, ChiBR96-LysPE40 on MCF-7 breast carcinoma cellsmeasured as inhibition of protein synthesis (FIG. 40). Determination ofinhibition of protein synthesis was as follows:

[0568] All cell lines were cultured as monolayers at 37° C. in RPMI 1640supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 50units/ml penicillin/streptomycin. Tumor cells were plated onto 96-wellflat bottom tissue culture plates (1×10⁴ cells/well) and kept at 37° C.for 16 hours. Dilutions of immunotoxin were made in growth media and 0.1ml added to each well for 20 hours at 37° C. Each dilution was done intriplicate. The cells were pulsed with [³H]-leucine (1 μCi/well) for anadditional 4 hours at 37° C. The cells were lysed by freeze-thawing andharvested using a Tomtec cell harvester (Orange, Conn.). Incorporationof [³H]-leucine was determined by a LKB Beta-Plate liquid scintillationcounter.

[0569] For competition experiments, tumor cells were prepared asdescribed above. BR96 IgG or, as a control, L6 IgG was diluted to 100μg/ml in growth media before addition to the cell monolayer (0.1ml/well). After incubation at 37° C. for 1 hour, dilutions of BR96sFv-PE40 were added, incubated an additional hour, cell supernatantswere removed, and cells were washed with complete RPMI growth media.Growth media (0.2 ml) was added to each well, cells were incubated at37° C. for 20 hours and were labelled with [³H]-leucine as describedabove.

[0570] The results indicate that the single-chain immunotoxin was 3-foldmore potent than the conjugate, with ID₅₀ values of 4 and 12 pM,respectively.

[0571] Next, in order to correlate the cytotoxicity with the presence ofthe BR96 antigen, the relative antigen density was determined on fivetumor cell lines by FACS analysis (FIG. 41). Assays were performed byfluorescence as described by Hellstrom et al., Cancer Res. 50:2183-2190(1990). Briefly, target cells were harvested in logarithmic phase withEDTA (0.02%) in calcium- and magnesium-free PBS. The cells were washedtwice in PBS containing 1% BSA and resuspended to 1×10⁷ cells/ml in PBScontaining 1% BSA and 0.02% NaN₃. Cells (0.1 ml) were mixed with BR96 ora human IgG control (0.2 ml at 50 μg/ml) and incubated for 45 minutes at4° C. The cells were washed 2 times and resuspended in 0.1 ml of anappropriate concentration of FITC labelled rabbit anti-human IgG(Cappel, Malvern, Pa.). Cells were incubated for 30 minutes at 4° C.,washed 2 times in PBS containing 0.02% NaN₃ and analyzed on a CoulterEPICS 753 fluorescence-activated cell sorter. Data are expressed as thefluorescence intensity of cells reacted with BR96 minus cells reactedwith control antibody. On a logarithmic scale, 25 units of fluorescenceintensity represents a doubling of antigen density. FACS analysis of anon-specific human IgG antibody was performed for each cell line todetermine non-specific fluorescence and a fluorescence intensity wascalculated (Table 11). TABLE 11 CYTOTOXICITY OF BR96 sFv-PE40 ON VARIOUSCELL LINES BR96 ID₅₀ Fluorescence ID₅₀ Cell Line Cancer Type Intensityng/ml (pM) MCF-7 Breast 177.8 0.3 (4.4) L2987 Lung 172.8 5.0 (75) RCAColon 138.5 8.0 (119) A2780 Ovarian 103.2 50.0 (750) KB Epidertnoid 33.6500.0 (7,462)

[0572] When the cytotoxic potential of BR96 sFv-PE40 was tested on thecell lines, it was found that inhibition of protein synthesis correlatedwith BR96 antigen density (Table 11). For example, MCF-7 cells were themost sensitive to BR96 sFv-PE40 (ID₅₀ of 4.4 pM) of the cell linestested. In contrast, KB cells which display negligible amounts of theBR96 antigen were much less sensitive to BR96 sFv-PE40 (ID₅₀ of 7,462pM). The cytotoxic activity of monomeric and aggregated BR96 sFv-PE40was also compared, and the monomer was demonstrated to be approximately50-60 times more effective at inhibiting protein synthesis than theaggregate population with ID₅₀ values on L2987 cells of 75 pM and 2920pM, respectively.

[0573] The competitive cytotoxicity experiments were conducted toconfirm the specificity of the immunotoxin for its antigen binding site(FIG. 42). The cytotoxic effect of BR96 sFv-PE40 is due to specificantigen binding, because the effect is severely reduced by excess BR96IgG, but not by L6 IgG, which does not recognize the BR96 antigen,.

[0574] Comparative Blood-Level Lifetime Analysis of BR96-Immunotoxins

[0575] BR96 sFv-PE40 is approximately one-third the size of theimmunotoxin conjugate, ChiBR96-LysPE40. Because protein size can affectbiological kinetics, the difference in blood half-life between BR96sFv-PE40 and ChiBR96-LysPE40 was measured. Both immunotoxins wereradioiodinated and administered to athymic mice via their tail veinusing the following procedures.

[0576] BR96 sFv-PE40 and ChiBR96-LysPE40 were labelled with Na ¹²⁵Iusing Chloramine T (McConahey et al., Arch. Allergy Appl. 29:185-188(1966)). Each reaction contained 100 μg of immunotoxin in PBS, 1 μCi ofNa ¹²⁵I, and 10 ng/ml chloramine T in a total reaction volume of 100 μl.

[0577] After a five minute incubation at room temperature, the reactionwas terminated by addition of 20 ng/ml Na-metabisulfide. The free Na¹²⁵I was separated from the radiolabeled immunotoxin by gel filtrationthrough PD-10 columns (Pharmacia). The specific activity of bothimmunotoxins was approximately 10 μCi/μg.

[0578] Female athymic mice (nu/nu) were purchased from Harlan SpragueDawley (Indianapolis, Ind.) at 4-6 weeks of age. The animals wereintravenously injected via the tail vein with 10 μCi of ¹²⁵I-BR96sFv-PE40 or ¹²⁵I-ChiBR96-LysPE40. The animal (2-4/data point) weresacrificed at various time points and the blood was collected andcounted in a gamma counter. The percent (%) ID for the blood wasdetermined as (CPM detected/CPM injected)×100. ID/ml was calculatedassuming a 1.6 ml total blood volume. Results are shown in Table 12.TABLE 12 SINGLE-CHAIN IMMUNOTOXIN VS. CHEMICAL CONJUGATE IMMUNOTOXINCOMPARATIVE BLOOD LEVEL ANALYSIS BR96 sFv-PE40 ChiBR96-LysPE40 Time %ID/ml Blood % ID/ml Blood  5 minutes 49.8 57.5 15 minutes 43.3 54.8 30minutes 28.2 46.3 60 minutes 15.5 41.5  2 hours 8.6 23.4  4 hours 5.222.0  6 hours 2.5 20.5 24 hours 0.2 7.2 48 hours 0.1 3.6

[0579] BR96 sFv-PE40 clears from the blood faster than ChiBR96-LysPE40.The estimated half-life in the blood for the single-chain immunotoxin isapproximately 30 minutes as compared to almost 2 hours for the chimericBR96 immunotoxin conjugate. In this experiment, the measurement¹²⁵I-labelled BR96 immunotoxin in the blood determined how much of themolecule was present.

[0580] In order to measure the amount of detectable single-chainimmunotoxin that was biologically active, the blood was assayed for BR96sFv-PE40 directed cytotoxic activity at the various times indicated inTable 12.

[0581] The results in this Example provide an expression plasmid for theproduction of a single-chain immunotoxin composed of thecarcinoma-reactive antibody of the invention, BR96 and a truncated formof Pseudomonas exotoxin. The chimeric molecule, BR96 sFv-PE40, purifiedfrom E. coli exists in both a monomeric and an aggregated form. Thespecificity of the monomeric BR96 sFv-PE40 for its antigen was confirmedthrough a competition analysis with BR96 IgG. The FACS analysis of fivedifferent cell lines demonstrates the distribution of the BR96 antigen,and the cytotoxic potential of BR96 sFv-PE40 was correlated with therelative number of antigen expressed on the surface of the target cells.BR96 sFv-PE40 is extremely potent against cancer cells displaying theBR96 antigen, with MCF-7 cells being the most sensitive cell lineexamined. BR96 sFv-PE40 was shown to be more potent than the BR96 IgGchemical conjugate against the tumor cell lines tested. To assess thepotential anti-tumor activity of BR96 sFv-PE40, the chimeric toxin wasintravenously administered to mice and found to have a serum half-lifeof 30 minutes, as compared to that of ChiBR96-LysPE40, which was almost2 hours. It may be an advantage that the single-chain immunotoxin iscleared so rapidly from the blood. The immunotoxin molecules are stableand retain biological activity following administration into animals.

EXAMPLE 15 In Vivo Effects of BR96 sFv-PE40

[0582] Anti-tumor Activity of BR 96 sFv-PE40 Against Human TumorXenographs L2987 and MCF-7 tumor fragments were implanted into femaleathymic mice (nu/nu) (Harlan Sprague Dawley, Indianapolis, Ind.) at 4-6weeks of age. They were implanted with L2987 and MCF-7 tumor fragmentsfrom established tumor xenografts that were approximately 4 weeks old(800 cu mm). Tumor sections were implanted subcutaneously using a trocaronto the back hind quarter of the mice. Two weeks after implantation theanimals were randomized and their tumors measured.

[0583] For the anti-tumor experiments, we only used animals that hadtumors ranging from 50-100 cubic mm in size. The animals wereintravenously injected via the tail vein with the BR96 sFv-PE40immunotoxin according to the administration schedule indicated in FIGS.43 and 44. Each treatment group consisted of five to ten animals.

[0584] Regression of MCF-7 breast carcinoma xenografts was observed withdoses up to 0.75 mg/kg using administration schedules of Q4DX3 (FIG.43). Using an administration schedule of Q2DX5, the L2987 lung tumorswere observed to regress upon treatment with BR96 sFv-PE40 (FIG. 44).Complete regression of the tumor xenografts was observed at dosesranging from 0.375 mg/kg to 0.125 mg/kg.

[0585] The effect of the tumor xenografts was dose-dependent, as atlower doses the tumors were able to grow back after being regressed fora seven day period while at higher doses the complete regression lastedfor over twenty days.

[0586] In untreated animals, the tumors grew rapidly and the animalswere sacrificed approximately 30 days post implantation. No apparenttoxicity was observed at the doses used in this experiment.

[0587] The tumor xenografts used in this study emanated from a smallpiece of a solid tumor excised from another animal. The tumor tissue wassubcutaneously implanted and allowed to vascularize and grow beforetreatment was initiated. In this manner, the data presented hereindemonstrate a tumor model of tumors found in humans.

EXAMPLE 16 Materials and Methods

[0588] Animals

[0589] Athymic mice and athymic Rowett rats (Harlan Sprague Dawley) wereused in this study.

[0590] The binding of BR96 to normal rat tissues was similar to BR96binding to normal human tissues, i.e., BR96 bound to cells in theesophagus, stomach, intestine and acinar cells of pancreas.

[0591] In contrast to rats, normal tissues from athymic mice did notbind BR96.

[0592] BR96-DOX

[0593] The conjugates were prepared by linking the DOX derivativemaleimidocaproyl doxorubicin hydrazone to BR96 or control immunoglobulin(FIG. 45). For more detail, see Examples 20, 22 and 26.

[0594] Implanted Carcinoma Cells

[0595] L2987 lung carcinoma cells were selected in vitro for the abilityto grow as multicellular spheroids. When injected IV into athymic miceor rats, tumors developed at various sites, including lymph nodes, lung,spleen, liver, brain, subcutaneously, and ascites was formed in someanimals.

[0596] Athymic rats were transplanted subcutaneously with human lungadenocarcinoma L2987, colon carcinoma RCA, or breast carcinoma MCF7 andpermitted to grow. Therapy (3 treatments 4 days apart) started 14 to 28days after tumor transplantation when tumors were well established,i.e., when the tumors were 50 to 100 mm³ in size.

[0597] Administration of BR96 into the Animals

[0598] Mice and rats were administered with BR96-DOX by threeinjections, each injection being about four days apart. Mice which wereinjected intraperitoneally (IP) were given 20 mg/kg of BR96-DOX. Micewhich were injected intravenously were given 10 mg/kg of BR96.Intravenous (IV) injection involved less volume of BR96 because of theconstraints of injection volume, namely, only 10 mg/kg was administrableby IV.

[0599] At the doses tested, there was no difference in the anti-tumoractivity of BR96-DOX whether administered IP or IV.

[0600] Controls One set of controls included untreated mice and micethat received (1) DOX (at doses optimized to produce maximal antitumoractivity in each model); (2) unconjugated BR96; (3) mixtures of BR96 andDOX; and (4) DOX conjugated to either normal human IgG or the controlMAb SN7. Doses of DOX and MAb are presented as mg/kg/infection.

[0601] Another set included rats using the protocol used in controlmice.

[0602] Results of Treatment of Mice

[0603] Treatment with BR96-DOX consistently cured most mice bearingL2987 (FIG. 46A) or RCA (FIG. 46B) tumors. Further, mice treated withBR96-DOX exhibited complete and partial tumor regressions against MCF7tumors (FIG. 46C). Complete tumor regression (CR) refers to a tumor thatfor a period of time is not palpable. Partial tumor regression (PR)means a decrease in tumor volume to ≦50% of the initial tumor volume.

[0604] Specifically, BR96-DOX cured 78% of the treated mice. Incontrast, DOX alone was not active against established RCA tumors eitherin terms of tumor growth delay or regressions.

[0605] The MTD of free DOX (4 mg/kg administered as 3 injections 4 daysapart) resulted in a delay in tumor growth and 25% cures. However,BR96-DOX given at a matching DOX dose (4 mg/kg DOX, 140 mg/kg BR96)cured 100% of the animals.

[0606] FIGS. 46A-D are line graphs showing the antigen-specificanti-tumor activity of BR96-DOX. FIG. 46A shows mice transplanted withL2987 lung tumor xenografts which have grown to about 50 to 100 mm³ atthe initiation of therapy. Treatment with BR96-DOX consistently curedmost mice bearing L2987 (FIG. 46A).

[0607]FIG. 46B shows mice transplanted with colon carcinoma RCA whichhave grown to tumor xenografts of about 50 to 100 mm³ at the initiationof therapy. Treatment with BR96-DOX consistently cured most mice bearingcolon carcinoma RCA (FIG. 46B).

[0608]FIG. 46C shows the efficacy of BR96-DOX in mice transplanted withMCF7 tumors. These mice treated with BR96-DOX consistently exhibitedcomplete and partial tumor regressions against MCF7 tumors (FIG. 46C).

[0609] Equivalent doses of non-binding IgG-DOX or SN7-DOX had no effectagainst these tumors. Although optimal doses of DOX delayed the growthof small L2987 tumors (50 to 100 mm³) and MCF7 tumors; regressions orcures were not observed.

[0610]FIG. 46D shows mice transplanted with L2987 lung tumor xenograftswhich grew to about 250 to 800 mm³ at the initiation of therapy. Suchmice which were treated with BR96-DOX also exhibited 56% cures, 22%complete and 22% partial regressions of lung tumors. In contrast,antitumor activity was not observed after treatment with an optimal doseof DOX.

[0611]FIG. 47 shows that BR96 is efficacious in curing athymic micehaving large disseminated tumors.

[0612] Mice were inoculated IV with L2987 spheroids. Approximatelytwelve weeks later mice (14 mice/group) were selected for treatment withBR96 (8 mg/kg equivalent DOX administered as 3 injections 4 days apart)or DOX on the basis of visible tumor burden, i.e., therapy was delayeduntil mice displayed extensive disseminated disease, ≧0.5g of visibletumor burden.

[0613] The burden of disseminated disease in these animals was so faradvanced that 50% of control animals died during the first 6 days of theexperiment. The median survival time (MST) of the control group was 90days and 100% of the mice were dead by day 102. Surviving mice weresacrificed 200 days after cell inoculation and sections of lung, lymphnodes, spleen, colon, jejunum, kidney, liver, brain, and heart wereexamined by histology.

[0614] Mice inoculated with L2987 spheroids and treated had an increasedMST (MST of >200 days) relative to that of control mice (MST of 85 days)or mice treated with an optimal dose of DOX (MST of 140 days).

[0615] According to immunohistology, the degree of binding of BR96 tocells from these carcinoma lines was similar to that of biopsy materialfrom human carcinomas of the same respective types (21).

[0616] Table 13 summarizes the tumor regression rates followingtreatment with various doses of (1) BR96-DOX, (2) DOX, and (3) mixturesof MAb and DOX against established L2987 and RCA tumor xenografts inmice.

[0617] BR96-DOX administered at equivalent DOX doses of ≧5 mg/kg (3injections 4 days apart) produced long-term cures in 72 to 100% of mice(n=291) bearing L2987 tumors.

[0618] In the RCA colon tumor model, which was not sensitive tounconjugated DOX, BR96-DOX administered at equivalent DOX doses of ≧10mg/kg (3 injections 4 days apart) cured 72 to 100% of mice (n=48).

[0619] Mice cured of L2987 or RCA tumors remained alive and tumor freefor more than 1 year with no indication of side effects. TABLE 13ANTITUMOR ACTIVITY OF BR96 − DOX AGAINST ESTABLISHED HUMAN TUMORXENOGRAFTS Dose (mg/kg/injection) PERCENT TUMOR REGRESSIONS No.:Treatment Schedule DOX MAb BR96* Tumor Cures Complete Partial MICE L2987BR96 − DOX q4dx3⁺ 20.0 689 100 0 0 8 15.0  711 ± 36 83.0 ± 0.8  3.3 ±0.9 7.0 ± 0.9 29 10.0  513 ± 12 83.0 ± 1.1  8.0 ± 0.7 2.0 ± 0.4 100 8.0317 ± 3 88.5 ± 0.1  3.7 ± 1.0 0 27 5.0 246 ± 5 72.3 ± 2.2 17.9 ± 1.5 5.6± 0.7 117 2.5 109 ± 3 30.4 ± 3.4 33.7 ± 2.4 21.3 ± 2.6  62 1.25  49 ± 1 6.9 ± 0.9 11.6 ± 1.2 11.9 ± 2.1  44 BR96 − DOX q1dx1≠ 30.0 1078  50.050.0 0 10 25.0 930 30.0 30.0 40.0 10 20.0 735 60.0 20.0 10.0 10 15.0 54011.0 22.0 44.0 9 IgG − DOX q4dx3 10.0   403 ± 5.2 0 0 0 19 5.0   202 ±3.2 0 0 3.7 ± 1.0 27 DOX q4dx3 8.0 — 0 0 0.8 ± 0.8 125 Mab BR96 q4dx3 —400 0 0 0 8 — 200 0 0 0 8 — 100 0 0 0 8 BR96 + DOX q4dx3 8.0 400 0 0 0 98.0 200 0 0 0 9 8.0 100 0 0 0 9 RCA BR96 − DOX q4dx3 20.0 903 100 0 0 1015.0 625 80.0 10.0 10.0 10 10.0   376 ± 5.4 71.7 ± 0.9 0 10.7 ± 0.1  285.0 176 11.0 22.0 11.0 9 2.5  90 0 0 5.5 ± 1.3 18 BR96 − DOX q7dx3@ 20.0900 100 0 0 10 15.0 625 100 0 0 10 10.0 420 80 10 0 10 5.0 210 10 0 1010 IgG − DOX q4dx3 10.0 405 0 0 0 10 DOX q4dx3 8.0 — 0 0 0 29 DOX q7dx310.0 — 0 0 0 10

[0620] BR96 administered at equivalent doses was not active againstestablished tumors (either in terms of tumor growth delay orregressions) and the tumor growth delay produced by mixtures of BR96 andDOX was equivalent to that of DOX administered alone.

[0621] Contrary to our expectations, cells lacking BR96 expression werenot detected after treatment with BR96-DOX. Also, cells obtained fromtumors that grew back after BR96-DOX induced regression were assensitive in vitro to DOX as the parental cell line.

[0622] The IC₅₀ (concentration required to produce 50% inhibition of³[H]-thymidine incorporation) was 0.4±0.1 μM and 0.3±0.2 μM DOX fortreatment and parental, respectively. These cells were also as sensitiveto BR96-DOX as the parental cell line with IC₅₀ values of 2.7±0.5 μM and2.6±0.8 μM equivalent DOX for parental and treated, respectively.

[0623] These data suggest that it may be possible to successfullyretreat tumors with several rounds of BR96-DOX therapy.

[0624] The maximum tolerated dose (MTD) (equivalent DOX dose) of theBR96-DOX conjugate (administered as 3 injections 4 days apart) was 20mg/kg administered intraperitoneally (IP). When administeredintravenously (IV) the MTD was ≧10 mg/kg. This was the maximum dose thatcould be administered IV because of the constraints of injection volume.

[0625] At the doses tested, there was no difference in the antitumoractivity of BR96-DOX whether administered IP or IV. At doses of BR96-DOX(Table 1) equivalent to ≧mg/kg of DOX (≧250 mg/kg BR96) more than 70% oftreated animals were cured of established L2987 tumors.

[0626] In fact, the BR96-DOX (Table 13) equivalent to ≧5 mg/kg of DOX(>250 mg/kg BR96) more than 70% of treated animals were cured ofestablished L2987 tumors. The BR96-DOX conjugate was active at doses aslow as 1 mg/kg equivalent DOX. Therefore, the BR96-DOX conjugate wasactive at a dose equivalent to {fraction (1/20)}th of its MTD.

[0627] These data demonstrate the broad range of therapeutic doses whichwere achieved with BR96-DOX. The MTD of unconjugated DOX (8 mg/kg IV and4 mg/kg IP) was lower than that of the BR96-DOX conjugate. UnconjugatedDOX administered IV at the MTD produced a delay in tumor growth but notumor regressions and if the dose was reduced to 50% of the MTD, DOX hadno effect.

[0628] In contrast, activity equivalent to that of an optimal dose ofDOX (8 mg/kg) was achieved at a dose of 1 mg/kg of BR96-DOX. TheBR96-DOX conjugate produced antitumor activity comparable to that of anoptimal dose of unconjugated DOX at ⅛th of the equivalent DOX dose. Insummary, the BR96-DOX conjugate was more active, had a much broaderrange of therapeutic doses, and was more potent than unconjugated DOX.

[0629] Seven of the 8 surviving mice were free of detectable tumor (70%cures by combined life span and histologic examination).

[0630] The BR96-DOX conjugate demonstrated strong antitumor activity inall preclinical models evaluated. The efficacy and potency of BR96-DOXconjugates is likely due to several factors. The antigen to which BR96binds is abundantly expressed at the tumor cell surface and active drugis released following antigen-specific binding and internalization ofthe conjugate into the acidic environment of lysosomes/endosomes.

[0631] Acid-labile immunoconjugates, in which a less stable disulfidelinker was used, have been investigated previously (7, 26). Althoughthese conjugates were active in an antigen-specific manner, they hadpoor potency in vivo (7). The use of a more stable thioether linker, anda MAb with higher avidity and more rapid rates of internalization,improved the activity and potency of BR96-DOX conjugates and alsoincreased the range of therapeutic doses.

[0632] We showed that administration of BR96-DOX conjugate at cumulativedoses of at least 15 mg/kg DOX and 700 mg/kg MAb (equivalent to 45 Mg/M²DOX and 2100 mg/m² MAb) resulted in greater than 70% cures ofestablished lung tumors. This dose of MAb in mice is approximatelyequivalent to a cumulative dose of 3 g of MAb per patient and is onlyslightly higher than that required to achieve saturation of humancarcinomas in patients given L6, another anticarcinoma MAb (G. Goodmanet al., J. Clin. Oncol., 8, 1083 (1990).

[0633] It would be clear to those skilled in the art that the optimalschedule for administering BR96-DOX will vary based upon the subject,the subject's height and weight, the severity of the disease.

[0634] The demonstration of tumor cures in animals in which BR96 bindsto normal tissues highlights the fact that the appropriate combinationof MAb, drug, and linker chemistry are critical aspects to successfulantibody-directed therapy. The toxic effects of DOX are dose related andit is likely that increasing the intra-tumoral concentration of DOX willproduce a significant increase in antitumor activity (S. K. Carter, J.Natl. Cancer Inst. 55, 1265 (1975); R. C. Young, R. F. Ozols, C. E.Myers, N. Eng. J. Med. 305, 139 (1981)).

[0635] BR96-DOX induced complete regressions and cures of xenograftedhuman lung, breast and colon carcinomas growing subcutaneously inathymic mice and cured 70% of mice bearing extensive metastases of ahuman lung carcinoma.

[0636] Results of Treatment with Rats The MTD of free DOX (4 mg/kgadministered as 3 injections 4 days apart) resulted in a delay in tumorgrowth and 25% cures.

[0637] The MTD of free DOX (4 mg/kg administered as 3 injections 4 daysapart) resulted in a delay in tumor growth and 25% cures. However,BR96-DOX given at a matching DOX dose (4 mg/kg DOX, 140 mg/kg BR96)cured 100% of the animals, and a dose equivalent to 2 mg/kg DOX (70mg/kg BR96) cured 88% of the rats.

[0638] BR96-DOX given at a matching DOX dose (4 mg/kg DOX, 140 mg/kgBR96) cured 100% of the animals, and a dose equivalent to 2 mg/kg GOX(70 mg/kg BR96) cured 88% of the rats. Of the rats treated withBR96-DOX, 94% (15/16) remained alive and tumor free with no evidence oftoxicity 150 days after the last dose of BR96-DOX.

[0639] It is surprising that BR96-DOX also cured 94% of athymic ratswith subcutaneous human lung carcinoma, even though the rats, likehumans, in contrast to mice, express the BR96 target antigen in somenormal tissues.

[0640] The BR96-DOX conjugate demonstrated antigen-specific activity invitro and was 8 to 25 fold more potent than non-binding (IgG-DOX orSN7-DOX) conjugates against carcinoma lines that expressed the BR96antigen. BR96-DOX was much less active against cells that did not bindBR96.

[0641] Optimal doses of DOX (8 mg/kg) had no effect on the largedisseminated tumors; the MST was 94 days and 100% of the mice were deadby day 140. In contrast, mice treated with BR96-DOX (8 mg/kg) had a MSTof ≧200 days and 8 of the 10 animals survived for the duration of theexperiment.

EXAMPLE 17 Conjugate of SPDP Thiolated Monoclonal Antibody BR64 with theMaleimidocaproyl Hydrazone of Adriamycin

[0642] A solution of the BR64 antibody (25 mL, 10.37 mg/mL; determinedby UV at 280 nm, 1.4 absorbance units equal 1 mg protein) was treatedwith SPDP solution in absolute ethanol (1.3 mL of 10 mmol solution). Thesolution was incubated for 1 hour at 31°-32° C., then chilled in ice andtreated with a solution of DTT in phosphate buffered saline (“PBS”) (1.3mL of a 50 mmol solution). The solution was kept in ice for 1 hour thentransferred to a dialysis tube and dialyzed three times against PBS (2 Lper dialysis) for a period of at least 8 hours. After the dialysis, theconcentration of protein was measured, as above, followed by adetermination of molar concentration of free sulfhydryl groups by theEllman method.

[0643] The thiolated protein (3 mL) was treated with an equivalent thiolmolar amount of maleimidocaproyl hydrazone of adriamycin, prepared as inPreparation 2, dissolved in dimethylformamide (DMF) (5 mg/mL, 0.131 mL)and the mixture was incubated at 4° C. for 24 hours. The solution wasdialyzed three times against PBS (1000 mL) for a period of at least 8hours. The solution was centrifuged and the supernatant was shaken for afew hours with Bio-beads™ SM-2 (non-polar neutral macroporouspolystyrene polymer beads, Bio-Rad Laboratories, Richmond, Calif. 94804)and finally filtered through a Millex-GV (Millipore Corporation,Bedford, Mass. 01730) 0.22 μm filter unit. The overall average number ofmolecules of adriamycin per molecule of antibody (“MR”) was determinedby measuring the amount of adriamycin from the absorption at 495 nm(ε=8030 cm⁻¹M⁻¹) and the amount of protein from the absorption at 280 nmafter correcting for the absorption of adriamycin at 280 nm according tothe formula:${{Antibody}\left( {{mg}\text{/}{mL}} \right)} = \frac{A_{280} - \left( {0.724 \times A_{495}} \right)}{1.4}$

[0644] The MR found for the product was 5.38; free adriamycin 0.14%;protein yield 60%.

EXAMPLE 18 Conjugate of SPDP Thiolated BR64 with the MaleimidocaproylHydrazone of Adriamycin

[0645] A solution of the BR64 antibody (405 mL, 11.29 mg/mL) was stirredand treated with SPDP solution in absolute ethanol (22.3 mL of 10 mmolsolution). The solution was incubated for 1 hour at 31°-32° C. whilebeing gently shaken, then cooled in ice to 4° C., stirred and treatedwith a solution of DTT in PBS (22.3 mL of a 50 mmol solution). Thesolution was kept in ice for 1 hour then divided into 2 equal parts,each transferred to a dialysis tube and dialyzed six times against PBS(6 L per dialysis) for a period of at least 8 hours. After that thecontents of the tubes were combined (400 mL) and the concentration ofprotein and free thiol groups was determined (molar ratio of —SH groupsto protein is 8.5).

[0646] The solution of thiolated protein was stirred and treated with anequivalent thiol molar amount of maleimidocaproyl hydrazone ofadriamycin dissolved in DMF (5 mg/mL, 35.7 mL) and the mixture wasincubated at 4° C. for 24 hours. The solution was divided into 2 equalparts, transferred to dialysis tubes and dialyzed five times against PBS(6 L per dialysis) for a period of at least 8 hours. The contents of thedialysis tubes were combined, filtered through a 0.22μ cellulose acetatefilter, and the filtrate was shaken for 24 hours with Bio-beads™ SM-2(Bio-Rad Laboratories, Richmond, Calif. 94804). the solution wasfiltered through a 0.22μ cellulose acetate filter. The concentration ofprotein and adriamycin was determined (6.26 mg/mL and 162.4 μg/mL,respectively) yielding a molar ratio (MR) of 7.18. The protein yield was77%. Unconjugated adriamycin present was 0.07%.

EXAMPLE 19 Conjugate of SPDP Thiolated SN7 with the MaleimidocaproylHydrazone of Adriamycin

[0647] In a manner analogous to that described in Examples 17 and 18,monoclonal antibody SN7, an antibody which does not bind to the antigenrecognized by BR64, was thiolated with SPDP and reacted with themaleimidocaproyl hydrazone of adriamycin to yield a conjugate with amolar ratio (MR) of 4. Protein yield was 51%. Unconjugated adriamycinpresent was 0.36%.

EXAMPLE 20 Conjugate of SPDP Thiolated CHiBR96 with the MaleimidocaproylHydrazone of Adriamycin

[0648] A solution of chimeric BR96 antibody, ChiBR96, (27.5 mL, 12.53mg/mL) was treated with a 10 mM solution of SPDP in absolute ethanol(1.7 mL). The solution was incubated at 31° C. for 35 minutes, chilledin ice and treated with a 0.50 mM solution of DTT in PBS (1.7 mL) for 15min at 4° C. The solution was transferred to a dialysis tube anddialyzed four times in PBS-0.1 M histidine buffer (4.5 L per dialysis)for a period of at least 8 hours. The amount of protein and molarconcentration of thiol groups was determined (9.29 mg/mL and 2.06×10⁻⁴M; respectively). The solution (17 mL) was treated with an equivalentmolar amount of the maleimidocaproyl hydrazone of adriamycin in DMF (5mg/mL, 0.59 mL) and the reaction mixture incubated at 4° C. for 24hours. The reaction mixture was dialyzed three times, in the same buffer(4.5 L per dialysis), for at least 8 hours. The dialyzed solution wascentrifuged and the supernatant shaken gently with BioBeads™ SM-2(Bio-Rad Laboratories, Richmond, Calif. 94804) for a few hours at 4° C.The solution was centrifuged and the concentration of protein andadriamycin in the supernatant (19 mL) was determined (6.5 mg/mL and67.86 μg/mL, respectively). The molar ratio of drug to protein is 2.9.Protein yield is 72%; unconjugated adriamycin present is 1.2%.

EXAMPLE 21 Conjugation of Modified Bombesin with the MaleimidocaproylHydrazone of Adriamycin

[0649] Bombesin does not contain a free reactive sulfhydryl group whichcan be used to link the drug through the Michael AdditionReceptor-containing linker. Thus, there was prepared a modified bombesinwhich contains an additional cysteine residue at the amino terminus ofnative bombesin. In addition, residue-3 of the native bombesin has beenchanged to a lysine residue. The modified bombesin, therefore, isdesignated “Cys⁰-lys³-bombesin.”

[0650] Cys⁰-lys³-bombesin (11.3 mg) was dissolved in 1.1 mL of deionizedwater and adjusted to pH 7-7.5 with 10 μl 1.5 M Tris-HCl, pH 8.8 andthen reacted with 0.45 mL maleimidocaproyl adriamycin hydrazone (15mg/mL in deionized water) at ambient temperature for several hours. Thereaction mixture was dialyzed against water overnight in dialysis tubing(molecular weight cutoff: 1000). The precipitate was removed bycentrifugation (12,000×g) and the supernatant was saved. Adriamycin(“ADM”) content of the bombesin-adriamycin conjugate was measured bydiluting 1:50 in acetate buffer, pH 6.0. The adriamycin (“ADM”) contentwas calculated using the formula:

[O.D.₄₉₅/8030]×50=ADM (M)

[0651] For this preparation O.D.₄₉₅=0.116 thus the adriamycin contentwas 7.2×10⁻⁴M.

[0652] The product was chromatographed by HPLC using a C₁₈ (BeckmanInstruments, Ultrasphere 5μl, 4.6 mm×25 cm) column. Buffer A: 10 mMNH₄OAc pH 4.5; Buffer B: 90% acetonitrile/10% Buffer A. The column wasequilibrated with 90% Buffer A/10% Buffer B and the chromatographyconditions were: 90% buffer A/10% buffer B to 60% Buffer A/60% buffer Bfor 2 minutes, gradient to 50% buffer A/50% buffer B for 15 minutes. Theproduct had a retention time of 9.3 minutes under these conditions.

EXAMPLE 22 A Conjugate of Iminothiolane Thiolated Chimeric BR96 andMaleimidocaproyl Hydrazone of Adriamycin

[0653] Chimeric BR96 (15 mL, 9.05 mg/mL) was dialyzed two times against4 liters of 0.1 M sodium carbonate/bicarbonate buffer, pH 9.1. Theantibody solution then was heated with iminothiolane (0.75 mL, 20 mM) at32° C. for 45 minutes. The solution was then dialyzed against 4 litersof sodium carbonate/bicarbonate buffer, pH 9.1 followed by dialysisagainst 4 liters of 0.0095 M PBS-0.1 M L-histidine, pH 7.4. Thissolution had a molar ratio of —SH/protein of 1.35. The protein then wasre-thiolated as described above to yield a solution with a molar ratioof —SH/protein of 5.0.

[0654] The maleimidocaproyl hydrazone of adriamycin (3.2 mg in 0.640 mLDMF) was added with stirring at 4° C. to the thiolated protein solution.The conjugate was incubated at 4° C. for 16 hours then it was dialyzedagainst 4 liters of 0.0095 M PBS-0.1 M L-histidine, pH 7.4. The dialyzedconjugate was filtered through a 0.22μ cellulose acetate membrane into asterile tube to which a small quantity (>5% (v/v)) of BioBeads™ SM-2(Bio-Rad Laboratories, Richmond, Calif. 94804) were added. After 24hours of gentle agitation, the beads were filtered off and the conjugatewas frozen in liquid nitrogen and stored at −80° C. The resultingconjugate had a molar ratio of 3.4 adriamycin molecules to 1 molecule ofprotein and was obtained in 24% yield from chimeric BR96.

EXAMPLE 23 Conjugate of Maleimidocaproyl Hydrozone of Adriamycin withDTT Reduced Human IgG (“Relaxes Human IgG”)

[0655] Human IgG (obtained from Rockland, Gilbertsville, Pa.) wasdiluted with 0.0095 M PBS to a protein concentration of 10.98 mg/mL.This solution (350 mL) was heated to 37° C. in a water bath under anitrogen atmosphere. Dithiothreitol (16.8 mL, 10 mM) in PBS was addedand the solution was stirred for 3 hours at 37° C. The solution wasdivided equally between two Amicon (Amicon Division of W. R. Grace andCo., Beverly, Mass. 01915) Model 8400 Stirred Ultrafiltration Cells,each fitted with an Amicon YM 30 Ultrafilter membrane (MW cutoff 30,000,76 mm diam.) and connected via an Amicon Model CDS10concentration/dialysis selector to an Amicon Model RC800 mini-reservoir.Each reservoir contained 700 mL of 0.0095 M PBS-0.1 M L-histidine. Theprotein solutions were dialyzed until concentration of free thoil in thefiltrate was 41 μM. the molar ratio of —SH/protein in the retentate wasdetermined to be 8.13.

[0656] The retentate was transferred from the cells to a sterilecontainer maintained under a nitrogen atmosphere and a solution ofmaleimidocaproyl hydrazone of adriamycin (36.7 mL, 5 mg/mL in water) wasadded with stirring. The conjugate was incubated at 4° C. for 48 hoursafter which it was filtered through a 0.22μ cellulose acetate membrane.A Bio-Rad Econocolumn™ (2.5 cm×50 cm, Bio-Rad Laboratories, Richmond,Calif. 94804) was packed with slurry of 100 g of BioBeads™ SM-2 (Bio-RadLaboratories, Richmond, Calif. 94804) in 0.00095 M-0.1 M L-histidinebuffer. The beads had been prepared by washing in methanol, followed bywater and then several volumes of buffer. The filtered conjugate waspercolated through this column at 2 mL/min. After chromatography theconjugate was filtered through a 0.22μ cellulose acetate membrane andfrozen in liquid nitrogen and stored at −80° C. The conjugate obtainedhad an average molar ratio of 7.45 molecules of adriamycin per moleculeof protein and was obtained in 99% yield from human IgG.

EXAMPLE 24 Conjugate of Relaxed BR64 with Maleimidocaproyl Hydrazone ofAdriamycin

[0657] A solution of BR64 (435 mL; 11.31 mg/mL, 7.07×10⁻⁵ M) was treatedwith DTT (947 mg) and heated at 42°-43° C. with gentle stirring for 2hours. The solution was cooled in ice, transferred into 2 dialysis tubesand each tube was dialyzed 5 times against PBS (14 L per dialysis) for 8hours at 4° C. The contents of the tubes were combined (400 mL) and theprotein and —SH content determined (10.54 mg/mL, 6.58×10⁻⁵ M, 5.14×10⁻⁴M, respectively). The molar ratio of —SH to protein was 7.8.

[0658] A solution of maleimidocaproyl hydrazone of adriamycin in DMF (5mg/mL, 32.6 mL) was added to the antibody solution with gentle stirringand then incubated at 4° C. for 24 hours. The solution was filteredthrough a 0.22μ cellulose acetate filter and then transferred to twodialysis tubes and dialyzed as described above. After dialysis, thecontents of the tubes were combined, filtered and shaken with BioBeads™SM-2 (Bio-Rad Laboratories, Richmond, Calif. 94804) for 24 hours at 4°C. The beads were filtered off using a cellulose acetate filter to yieldthe conjugate solution. The concentration of protein and adriamycin weredetermined (8.66 mg/mL, 5.42×10⁻⁵ M; 168 μg/mL, 2.89×10⁻⁴ M,respectively). The protein yield is 97%. The molar ratio of adriamycinto protein is 5.33; and, unconjugated adriamycin is 0.5%.

EXAMPLE 25 General Procedure for Conjugating the MaleimidocaproylHydrazone of Adriamycin to a Relaxed Antibody

[0659] 1. A solution (300 mL) of antibody (3 g, 10 mg/mL) in BPS buffer(note 1) is continuously blanketed with nitrogen, immersed in a 37° C.water bath and stirred gently with a magnetic stirrer. The solution istreated with 7 molar equivalents of DTT (notes 2, 3) for 3 hours. The—SH group molar ratio (“MR”) to protein is determined initially andhourly and, for a maximally conjugated product, should remain constantat about 14 (notes 2, 4).

[0660] 2. The solution is transferred as quickly as possible to anAmicon diafiltration cell (Amicon, Division of W. R. Grace and Co.,Beverly, Mass. 01915) (note 5) maintained at about 4° C. to about 7° C.The system is pressurized with argon or nitrogen and diafiltration isstarted using PBS buffer containing 0.1 M histidine which has beenprecooled to about 4° C. to about 7° C. The initial temperature of theeffluent, immediately after starting the diafiltration, is 16°-18° C.and drops to 8°-9° C. within about 90 minutes. The effluent is monitoredfor a MR of —SH to protein and, when this value is <1, the diafiltrationis complete (note 6).

[0661] 3. The solution is transferred back to a round bottom flaskequipped with a magnetic stirrer and kept in ice. The solutioncontinuously is blanketed by nitrogen. The volume of the solution isnoted. Aliquots of 0.1 mL are taken out and diluted with PBS buffer to1.0 mL to determine the amount of protein in mg/mL (and also the molarequivalent of protein and the molarity of the —SH groups (and hence theMR of the —SH to protein)). A solution of maleimidocaproyl hydrazone ofadriamycin in distilled water (5 mg/mL, 6.3×10⁻³ M) is prepared (notes7, 8). The amount (in mL) of this solution needed for the conjugation isdetermined by the formula: $\frac{\begin{matrix}{\text{(molarity of}\text{-}\text{SH)} \times} \\{\text{(volume~~of~~protein~~solution)} \times 1.05}\end{matrix}}{6.3 \times 10^{- 3}}$

[0662] (note 9) and this amount is added slowly to the protein solutionwhich is stirred gently. The solution is kept at 4° C. for 30 minutes.

[0663] 4. A column of BioBeads™ SM-2, mesh 20-50 (Bio-Rad Laboratories,Richmond, Calif. 94804) is prepared (note 10) at 4° C. The red proteinsolution is filtered through a 0.22μ cellulose acetate filter, thenpassed through the column at a rate of 2.5 mL/min and the red effluentcollected. Finally PBS-0.1 M histidine buffer is poured on top of thecolumn and the effluent collected until it is colorless. The volume ofthe collected red solution is noted. An aliquot of 0.1 mL is diluted to1 mL with PBS buffer and the amount of protein and adriamycin ismeasure. The amount of conjugated adriamycin is determined by absorbanceat 495 nm (ε=8030 cm⁻¹M⁻¹) and expressed in micromoles and microgramsper mL. The amount of protein, expressed in mg per mL and micromoles, isdetermined as above by reading the absorbance at 280 nm with acorrection for the absorbance of adriamycin at the same wavelengthaccording to the general formula:${{Antibody}\left( {{mg}\text{/}{mL}} \right)} = \frac{A_{280} - \left( {0.724 \times A_{495}} \right)}{1.4}$

[0664] where A is the observed absorbance at the noted wavelength. TheMR of adriamycin to protein then is calculated.

[0665] 5. An aliquot of 5 mL of conjugate is passed over an Econo-Pac™10 SM-2 column (a prepacked Bio-Beads™ SM-2 column (Bio-RadLaboratories, Richmond, Calif. 94804), volume 10 mL, that has beenwashed and equilibrated with PBS-0.1 M histidine buffer) in the mannerdescribed above. The amount of protein and conjugated adriamycin isdetermined and the MR determined. This value should be the same as thatof the bulk of the solution (note 11).

[0666] 6. The conjugate is frozen in liquid nitrogen and stored at −80°C. Aliquots can be taken for determining cytotoxicity, binding andpresence of free adriamycin (note 12) concentration by the molar proteinconcentration. Should this value be less than 14 during the reaction anappropriate additional amount of DTT is added.

[0667] 7. On a scale of 3 g/300 mL, two Amicon cells of 350 mL each areused, dividing the solution into two portions of 150 mL per cell.

[0668] 8. On the reaction scale provided, the diafiltration usuallytakes 2-4 hours. The duration will depend on factors such as the age ofthe membrane, rate of stirring of solution and pressure in cell.

[0669] 9. The hydrazone is not very soluble in PBS and a precipitate isformed in a short while.

[0670] 10. Brief applications of a sonicator will facilitate dissolutionin distilled water. The resulting solution is stable.

[0671] 11. This amount provides for a 5% excess of the hydrazone. Theprocess described generally takes about 15-20 minutes.

[0672] 12. The Bio-Beads™ are prepared for chromatography by swellingthem in methanol for at least one hour, preferably overnight, washingthem with distilled water and finally equilibrating them with PBS-0.1 Mhistidine buffer. For 3 g of protein 100 g of beads are used to form acolumn of 2.5 cm×30 cm.

[0673] 13. Because of the inherent error of the spectroscopic methodsused, a variation of 1 MR unit is accepted to be a satisfactory result.Generally, however, the MR varies less than 0.5 MR units.

[0674] 14. The values of free adriamycin in the conjugate are generallymuch less than 1%.

EXAMPLE 26 Conjugate of Relaxed Chimeric BR96 with MaleimidocaproylHydrazone of Adriamycin

[0675] Chimeric BR96, prepared in the manner previously described, wasdiluted with 0.0095 M PBS to a protein concentration of 10.49 mg/mL.This solution (500 mL) was heated to 37° C., under a nitrogenatmosphere, in a water bath. Dithiothreitol (26.2 mL, 10 mM) in PBS wasadded and the solution was stirred for 3 hours at 37° C. The solutionwas divided equally between two Amicon Model 8400 stirredultrafiltration cells each fitted with a YM 30 ultrafilter (MW cutoff30,000, 76 mm diam.) and connected via a Model CDS10concentration/dialysis selector to a Model RC800 mini-reservoir (Amicon,Division of W. R. Grace and Co., Beverley, Mass. 01915). Each reservoircontained 800 mL of 0.0095 M PBS-0.1 M L-histidine. The proteinsolutions were dialyzed until the concentration of free thiol in thefiltrate was 60 μM. The molar ratio of —SH/protein in the retentate wasdetermined to be 8.16. The retentate was transferred from the cells to asterile container under nitrogen and a solution of maleimidocaproylhydrazone of adriamycin (42.6 mL, 5 mg/mL in water) was added withstirring. The conjugate was incubated at 4° C. for 48 hours after whichit was filtered through a 0.22μ cellulose acetate membrane. A 2.5 cm×50cm Bio-Rad Econocolumn was packed with slurry of 100 g of BioBeads™ SM-2(Bio-Rad Laboratories, Richmond, Calif. 94804) in 0.00095 M-0.1 ML-histidine buffer. The beads had been prepared by washing in methanol,followed by water then several volumes of buffer. The filtered conjugatewas percolated through this column at 2 mL/min. After chromatography theconjugate was filtered through a 0.22μ cellulose acetate membrane,frozen in liquid nitrogen and stored at −80° C. The conjugate obtainedhad a molar ratio of 6.77 adriamycin to protein and was obtained in 95%yield from chimeric BR96.

EXAMPLE 27 Conjugate of Relaxed Murine Antibody L6 with MaleimidocaproylHydrazone of Adriamycin

[0676] Murine antibody L6, prepared as defined earlier, was diluted with0.0095 M PBS to a protein concentration of 11.87 mg/mL. This solution(350 mL) was heated to 37° C., under a nitrogen atmosphere, in a waterbath. Dithiothreitol (18.2 mL, 10 mM) in PBS was added and the solutionwas stirred for 3 hours at 37° C. The solution was divided equallybetween two Amicon Model 8400 stirred ultrafiltration cells each fittedwith a YM 30 ultrafilter (MW cutoff 30,000, 76 mm diam.) and connectedvia a Model CDS10 concentration/dialysis selector to a Model RC800min--reservoir (Amicon, Division of W. R. Grace and Co., Beverly, Mass.01915). Each reservoir contained 800 mL of 0.0095 M PBS-0.1 ML-histidine. The protein solutions were dialyzed until concentration offree thiol in the filtrate was 14 μM. The molar ratio of —SH/protein inthe retentate was determined to be 9.8. The retentate was transferredfrom the cells to a sterile container under nitrogen and a solution ofmaleimidocaproyl hydrazone of adriamycin (40.4 mL, 5 mg/mL in water) wasadded with stirring. The conjugate was incubated at 4° C. for 48 hoursafter which it was filtered through a 0.22μ cellulose acetate membrane.A 2.5 cm×50 cm Bio-Rad Econocolumn was packed with a slurry of 100 g ofBioBeads™ SM-2 (Bio-Rad Laboratories, Richmond, Calif. 94804) in 0.00095M-0.1 M L-histidine buffer. The beads had been prepared by washing inmethanol, followed by water then several volumes of buffer. The filteredconjugate was percolated through this column at 2 mL/min. Afterchromatography the conjugate was filtered through a 0.22μ celluloseacetate membrane, frozen in liquid nitrogen and stored at −80° C. Theconjugate obtained had a molar ratio of 7.39 Adriamycin to protein andwas obtained in 100% yield from murine L6.

BIOLOGICAL ACTIVITY

[0677] Representative conjugates of the present invention were tested inboth in vitro and in vivo systems to determine biological activity. Inthese tests, the potency of conjugates of cytotoxicity drugs wasdetermined by measuring the cytotoxicity of the conjugates against cellsof human cancer origin. The following describes representative testsused and the results obtained. Throughout the data presented, theconjugates are referred to using the form ligand-drug-molar ratio ofligand to drug. Thus, for example, “BR64-ADM-5.33” refers to a conjugatebetween antibody BR64 and adriamycin and the mole ratio of drug toantibody is 5.33. One skilled in the art will recognize that any tumorline expressing the desired antigen could be used in substitution of thespecific tumor lines used in the following analyses.

TEST I In Vitro Activity OF BR64-Adriamycin Conjugates

[0678] The immunoconjugates of Examples 18 and 19 were tested in vitroagainst a human lung carcinoma line, L2987 (obtained from I. Hellström,Bristol-Myers Squibb Seattle; see also I. Hellström, et al., CancerResearch 50:2183 (1990)), which expresses the antigens recognized bymonoclonal antibodies BR64, L6 and BR96. Monolayer cultures of L2987cells were harvested using trypsin-EDTA (GIBCO, Grand Island, N.Y.), andthe cells counted and resuspended to 1×10⁵/mL in RPMI-1640 containing10% heat inactivated fetal calf serum (“RPMI-10% FCS”). Cells (0.1mL/well) were added to each well of 96-well flat bottom microtiterplates and incubated overnight at 37° C. in a humidified atmosphere of5% CO₂. Media was removed from the plates and serial dilutions ofadriamycin or the antibody conjugates of adriamycin were added to thewells. All dilutions were performed in quadruplicate. Following a 2 hourdrug or conjugate exposure, the plates were centrifuged (100×g, 5 min.),the drug or conjugate removed, and the plates washed three times withRPMI-10% FCS. The cells were cultured in RPMI-10% FCS for an additional48 hours. At this time the cells were pulsed for 2 hours with 1.0μCi/well of ³H-thymidine (New England Nuclear, Boston, Mass.). Theplates were harvested and the counts per minute (“CPM”) determined.Inhibition of proliferation was determined by comparing the mean CPM fortreated samples with that of the untreated controls. The data presentedin FIG. 51 demonstrates the cytotoxicity against L2987 lung cells ofbinding immunoconjugate (MR of adriamycin to BR64 equal to 7.18,designated BR64-THADMHZN-7.18“) compared to a non-bindingimmunoconjugate of SN7 and adriamycin (MR of adriamycin to SN7 equal to4, designated “SN7-THADMHZN-4”). The BR64 conjugates prepared by themethod described in Example 18 are active and demonstrateantigen-specific cytotoxicity in this in vitro screen.

TEST II In Vivo Activity of BR64-Adriamycin Conjugates

[0679] The immunoconjugates of Examples 18 and 19 were evaluated in vivofor antigen-specific antitumor activity. Congenitally athymic femalemice of BALB/c background (BALB/c nu/nu; Harlan Sprague-Dawley,Indianapolis, Ind.) were used in these studies. Mice were housed inThoren caging units on sterile bedding with controlled temperature andhumidity. Animals received sterile food and water ad libitum. The L2987human lung tumor line, described above, was used in these studies. Thisline has been shown to maintain expression of the BR64, BR96 and L6antigens following repeated passage in vivo. The tumor lines weremaintained by serial passage in athymic mice as described previously (P.A. Trail, et al., in vivo 3 319-324 (1989)). Tumors were measured, usingcalipers, in 2 perpendicular directions at weekly or biweekly intervals.

[0680] Tumor volume was calculated according to the equation:${V\left( {mm}^{3} \right)} = \frac{\left( {L \times W^{2}} \right)}{2}$

[0681] in which

[0682] V=volume (mm³)

[0683] L=measurement of longest axis (mm)

[0684] W=measurement (mm) of axis perpendicular to L

[0685] Data are presented as the median tumor size for treated andcontrol groups. Each treatment or control group contained 8-10 animals.Therapy was initiated when tumors had reached a median size of 50-100mm³. Therapy was administered by the ip or iv route on various schedulesas denoted. Adriamycin was diluted in normal saline and native antibodyand adriamycin conjugates were diluted in phosphate buffered saline(“PBS”) for administration. All dosages were administered on a weightbasis (mg/kg) and were calculated for each animal. In these studies theantitumor activity of binding BR64 immunoconjugates was compared to thatof optimized dosages of adriamycin, mixtures of native BR64 andadriamycin, and non-binding conjugates. Unconjugated adriamycin wasadministered according to the route, dosage, and schedule demonstratedto be optimal for the L2987 human xenograft model. The unconjugatedadriamycin, therefore, was administered at a dose of 8 mg/kg by the ivroute every fourth day for a total of 3 injections (denoted “8 mg/kg,q4dx3, iv”). The binding (BR64) and non-binding (SN7) immunoconjugateswere administered at several doses by the ip route every fourth day fora total of 3 injections (denoted “q4dx3, ip”). As shown in FIG. 52,significant antitumor activity was observed following the administrationof tolerated doses (10 and 15 mg/kg/injection) of the BR64-adriamycinconjugate. The antitumor activity observed following therapy with theBR64 conjugate was significantly better than that observed for therapywith optimized adriamycin and matching doses of non-binding (SN7)conjugate.

[0686] In this experiment, complete tumor regressions were observed in66% of the animals following treatment with 15 mg/kg/injection of theBR64 conjugate and 50% complete tumor regressions were observedfollowing treatment with 10 mg/kg/injection of the BR64 conjugate.Partial or complete regressions of established L2987 tumors have notbeen observed following therapy with optimized adriamycin, mixtures ofnative BR64 and adriamycin, or equivalent doses of non-bindingconjugates.

[0687] To demonstrate that the observed activity required the covalentcoupling of the antibody to adriamycin, several control experimentsusing mixtures of native BR64 and adriamycin were performed.Representative data for several types of combined therapy are shown inFIGS. 5a-c. The antitumor activity observed for various modes ofcombined therapy with MAb and adriamycin was not significantly differentfrom that observed for therapy with optimized adriamycin alone. Takentogether these data indicate that the covalent coupling of BR64 toadriamycin is required to observe the antitumor activity described inFIG. 21.

TEST III In Vivo Activity of Bombesin Conjugates

[0688] The conjugate of Example 21 was evaluated in vivo for antitumoractivity. BALB/c athymic nude mice were implanted with H345 human smallcell lung carcinoma tumor pieces (obtained from Dr. D. Chan, Universityof Colorado Medical School, Colo.), subcuntaneously, using trocars.Tumors were allowed to grow to 50-100 mm³ before initiation oftreatment. Mice were treated i.v. on 23, 26, 28, and 30 dayspost-implant with adriamycin alone (1.6 mg/kg), or the conjugatesbombesin-adriamycin (“BN-ADM(TH)”, in an amount equivalent to 1.6 mg/kgadriamycin) or P77-adriamycin conjugate (P77-ADM (TH)”, in an amountequivalent to 1.6 mg/kg of adriamycin). P77 is a 12 amino acid peptidewith an internal cysteine residue (sequence=KKLTCVQTRLKI) that does notbind to H345 cells and was conjugated to the maleimidocaproyl hydrazoneof adriamycin according to the procedure outlined in Example 21. Thus,the conjugate represents a non-binding conjugate with respect to H345cells. Tumors were measured with calipers and tumor volume wascalculated using formula:${V\left( {mm}^{3} \right)} = \frac{\left( {L \times W^{2}} \right)}{2}$

[0689] in which V, L, and W are as defined in Test II.

[0690] The median tumor volumes were determined and the observed resultsare shown in FIG. 54.

TEST IV In Vitro Cytotoxicity Data for Relaxed ChiBR96 AntibodyConjugates

[0691] Immunoconjugates of adriamycin and ChiBR96 antibody are preparedusing the general method for preparing relaxed antibodies as describedin Example 25. The conjugates were tested, using the protocol below, forin vitro cytotoxicity and their cytotoxicity was compared to that offree adriamycin, and SPDP-thiolated immunoconjugates prepared by themethod described in Example 18. The results of these tests are providedin FIG. 55.

[0692] Monolayer cultures of L2987 human lung cells were maintained inRPMI-1640 media containing 10% heat inactivated fetal calf serum(RPMI-10% FCS). The cells were harvested using trypsin-EDTA (GIBCO,Grand Island, N.Y.), and the cells counted and resuspended to 1×10⁵/milin RPMI-10% FCS. Cells (0.1 ml/well) were added to each well of 96 wellmicrotiter plates and incubated overnight at 37° C. in a humidifiedatmosphere of 5% CO₂. Media was removed from the plates and serialdilutions of adriamycin or antibody/ADM conjugates added to the wells.All dilutions were performed in quadruplicate. Following a 2 hour drugor conjugate exposure, the plates were centrifuged (200×g, 5 min.), thedrug or conjugate removed, and the plates washed 3× with RPMI-10% FCS.The cells were cultured in RPMI-10% FCS for an additional 48 hours. Atthis time the cells were pulsed for 2 hours with 1.0 μCi/well of³H-thymidine (New England Nuclear, Boston, Mass.). The plates wereharvested and the counts per minute (“CPM”) were determined. Inhibitionof proliferation was determined by comparing the mean CPM for treatedsamples with that of the untreated control. IC₅₀ values are reported asμM of equivalent adriamycin.

TEST V In Vivo Antitumor Activity of BR64 and Murine L6 Conjugates

[0693] The in vivo antitumor activity of immunoconjugates of adriamycinand relaxed BR64 or relaxed L6 was evaluated. The observed data areprovided in FIG. 56.

[0694] Congenitally athymic female mice of BALB/c background (BALB/Cnu/nu; Harlan Sprague-Dawley, Indianapolis, Ind.) were used. Mice werehoused in Thoren caging units on sterile bedding with controlledtemperature and humidity. Animals received sterile food and water adlibitum.

[0695] The L2987 human tumor line was established as tumor xenograftmodels in athymic mice. The tumor line was maintained by serial passagein vivo. Tumors were measured in 2 perpendicular directions at weekly orbiweekly intervals using calipers. Tumor volume was calculated accordingto the equation:${V\left( {mm}^{3} \right)} = \frac{\left( {L \times W^{2}} \right)}{2}$

[0696] in which:

[0697] V=volume (mm³)

[0698] L=measurement of longest axis (mm)

[0699] W=measurement of axis perpendicular to L

[0700] In general, there were 8-10 mice per control or treatment group.Data are presented as median tumor size for control or treated groups.Antitumor activity is expressed in terms of gross log cell kill (“LCK”)where: ${LCK} = \frac{T - C}{3.3 \times {TVDT}}$

[0701] T−C is defined as the median time (days) for treated tumors toreach target size minus the median time for control tumors to reachtarget size and TVDT is the time (days) for control tumors to double involume (250-500 mm³). Partial tumor regression (“PR”) refers to adecrease in tumor volume to ≦50% of the initial tumor volume; completetumor regression (“CR”) refers to a tumor which for a period of time isnot palpable; and cure is defined as an established tumor which is notpalpable for a period of time ≧10 TVDTs.

[0702] For animals bearing the L2987 human lung tumor, therapy wastypically initiated when the median tumor size was 75 mm³(12-14 daysafter tumor implant). The average TVDT was 4.8±0.9 days and antitumoractivity was assessed at a tumor size of 500 mm³. In several experiments(described below in Test VI) therapy was initiated when L2987 tumorswere 225 mm³ in size.

[0703] Materials under investigation were administered by the ip or ivroute. Adriamycin was diluted in normal saline; antibody andantibody/adriamycin conjugates were diluted in phosphate bufferedsaline. Compounds were administered on a mg/kg basis calculated for eachanimal, and doses are presented as mg/kg of equivalentadriamycin/injection. Immunoconjugates were administered on a q4dx3schedule. The maximum tolerated dose (“MTD”) for a treatment regimen isdefined as the highest does on a given schedule which resulted in ≧20%lethality.

[0704] In the data shown in FIG. 56, injection of optimized doses ofadriamycin produced antitumor activity equivalent to 1.1 LCK and tumorregressions were not observed. The BR64-ADM conjugate produced antitumoractivity equivalent to >10 LCK at all doses tested and 89%, 78%, and100% cures were observed at doses of 5 mg/kg, 8 mg/kg, and 10 mg/kg ofBR64-ADM, respectively. At doses of 8 mg/kg or 10 mg/kg the L6-ADMconjugate produced antitumor activity (1.8 and 3.5 LCK, respectively)which was significantly better than that of optimized adriamycin butless than that of equivalent doses of internalizing BR64-ADM conjugates.Thus, the data show that the antitumor activity of bindingnon-internalizing L6-ADM conjugates is superior to that of unconjugatedadriamycin. Treatment with L6-adriamycin conjugate results in lowerantitumor activity than is observed with matching doses of theinternalizing BR64-adriamycin conjugate.

TEST VI In Vivo Antitumor Activity of ChiBR96-ADM Conjugates

[0705] The antitumor activity of ChiBR96-ADM conjugates was evaluatedagainst established human lung (“L2987”), breast (“MCF7,” obtainablefrom the ATCC under the accession number ATCC HTB 22; see also I.Hellström, et al., Cancer Research 50:2183 (1990)), and colon (“RCA”from M. Brattain, Baylor University; see also I. Hellström, et al.,Cancer Research 50:2183 (1990)) tumors.

[0706] Animals were maintained and tumor xenograft models wereestablished for the MCF7 and RCA and the L2987 human tumor lines asdescribed for the L2987 in Test V. Materials were administered asdescribed in Test V.

[0707] For animals bearing the L2987 human lung tumor, therapy typicallywas initiated when the median tumor size was 75 mm³ (12-14 days aftertumor implant). The average TVDT was 4.8±0.9 days and antitumor activitywas assessed at a tumor size of 500 mm³. In several experiments therapywas initiated when L2987 tumors were 225 mm³ in size.

[0708] The MCF7 tumor is an estrogen-dependent human breast tumor line.Athymic mice were implanted with 0.65 mg (65 day release rate) estradiolpellets (Innovative Research of America, Toledo, Ohio) on the day oftumor implant. Therapy was initiated when the median tumor size was 100mm³ (typically 13 days after tumor implant). The MCF7 tumor had anaverage TVDT of 6.4±2.0 days and antitumor activity was assessed at 400mm³.

[0709] For animals bearing the RCA colon tumor, therapy was initiated 15days after tumor implant when the median tumor size was 75 mm³. Theaverage TVDT for RCA tumor xenografts was 9.5±1.5 days and antitumoractivity was assessed at 400 mm³. Data for the antitumor activity ofoptimized adriamycin in the L2987, MCF7, and RCA xenograft models issummarized in the following Tables and referenced Figures.

[0710] The antitumor activity of the ChiBR96-ADM conjugates was comparedto that of optimized adriamycin and equivalent doses of non-binding(IgG) immunoconjugates. In each model, complete tumor regressions and/orcures of established tumors were observed following the administrationof tolerated doses of ChiBR96-ADM conjugate.

[0711] Representative data demonstrating the antigen-specific antitumoractivity of ChiBR96-ADM conjugates is presented in FIGS. 57 and 48. Asshown in FIG. 57, the ip administration of ChiBR96-ADM conjugate(MR=4.19) at a dose of 10 mg/kg equivalent of adriamycin producedantitumor activity equivalent to >10 LCK. At this does of ChiBR96-ADMconjugate, 78% of the mice were cured of the tumor and an additional 11%of the mice demonstrated a complete tumor regression. The administrationof 5 mg/kg of the ChiBR96-ADM conjugate also produced antitumor activityequivalent to >10 LCK with 88% tumor cures and 12% complete tumorregressions. The antitumor activity observed following administration ofChiBR96-ADM conjugates (>10 LCK) was substantially better than thatobserved for optimized adriamycin (1.0 LCK). The ChiBR96-ADM conjugatewas also more potent than optimized adriamycin; that is, the antitumoractivity of the ChiBR96-ADM conjugate tested at a dose of 5 mg/kgequivalent adriamycin was superior to that of adriamycin tested at adose of 8 mg/kg. The non-binding human IgG conjugate (MR=7.16) was notactive against L2987 xenografts when tested at a dose of 10 mg/kgequivalent of adriamycin indicating that the superior activity of theChiBR96-ADM conjugate was due to antigen specific binding of theimmunoconjugate to L2987 tumor cells.

[0712] Similar data are presented in FIG. 58. As shown, the ChiBR96-ADMconjugate (MR=5.8) tested at a dose equivalent of 10 mg/kg adriamycinresulted in antitumor activity equivalent to >10 LCK. At this dose, 90%tumor cures and 10% complete tumor regressions were observed. Theadministration of 5 mg/kg of the ChiBR96-ADM conjugate resulted in 4.8LCK with 10% cures, 50% complete and 10% partial tumor regressions. Theantitumor activity of ChiBR96-ADM conjugate greatly exceeded that ofoptimized adriamycin (1.6 LCK) and, as described above, the ChiBR96-ADMconjugate was more potent than unconjugated adriamycin. The non-bindingIgG-ADM conjugate (MR=7.16) was not active at a dose of 10 mg/kg.

[0713] The antitumor activity of various preparations of ChiBR96-ADMconjugates prepared by the “relaxed” antibody technique and evaluatedagainst established L2987 lung tumor xenograft is presented in Table 15.TABLE 15 Antitumor Activity of ChiBR96-ADM Conjugates AgainstEstablished L2987 Human Lung Tumor Xenografts* Dose (mg/kg) % TumorRegressions Conjugate ADM Antibody Route LCK PR CR Cure No. of MiceChiBR96-ADM-6.85 15 615 ip >10 10 0 80 10 10 410 ip >10 0 0 89 9 8 328iv >10 0 0 100 9 5 205 iv >10 0 22 78 9 ChiBR96-ADM-4.19 15 980 ip >10 011 89 9 10 654 ip >10 11 11 66 9 5 327 iv >10 0 11 89 9 2.5 164 iv >10 022 78 9 ChiBR96-ADM-6.85 10 410 ip >10 11 11 78 9 8 328 iv >10 0 0 100 95 205 iv >10 0 11 89 9 ChiBR96-ADM-4.19 10 654 ip >10 0 0 100 9 5 327iv >10 0 0 100 9 ChiBR96-ADM-4.19 10 654 ip >8 0 22 78 9 5 327 ip >8 011 89 9 ChiBR96-ADM-5.80 10 500 ip >10 0 10 90 10 5 250 ip >4.8 10 50 1010 ChiBR96-ADM-6.82 5 204 iv >10 22 22 55 9 2 82 iv 3.5 44 33 0 9 1 41iv 2.0 0 22 0 9 ChiBR96-ADM-6.82 10 400 ip >5.3 11 11 56 9 5 200 ip 4.830 10 40 10 2.5 100 ip 2.9 30 0 30 10 1.25 50 ip 1.1 11 0 11 9 0.62 25ip 0 0 0 0 9 5 200 iv >5.3 10 20 70 10 2.5 100 iv 2.9 22 33 0 9 1.25 50iv 1.5 11 11 0 9 0.62 25 iv 0.6 0 0 0 9 Adriamycin 8 — iv 1-1.8 3.6 0 055

[0714] TABLE 17 Summary of Antitumor Activity of ChiBR96-ADM ThioetherConjugates Evaluated Against Established MCF7 Human Breast TumorXenografts Dose (mg/kg)^(a) % Tumor Regressions Conjugate ADM ChiBR96Route LCK PR CR Cure No. of Mice ChiBR96-ADM-7.88 10 350 ip —^(b) — — —10 5 175 ip 4.2 30 0 0 10 5 175 iv 4.2 50 10 0 10 IgG-ADM-7.16 5 225 ip1.1 0 0 0 10 2.5 112 ip 0.6 0 0 0 10 2.5 112 iv 0.8 0 0 0 10 Adriamycin6 0 iv 1.4 0 0 0 10

[0715] As shown, the antitumor activity of ChiBR96-ADM conjugates issuperior to that of optimized adriamycin and the ChiBR96-ADM conjugatesare 6-8 fold more potent than unconjugated adriamycin.

[0716] The antitumor activity of ChiBR96-ADM conjugates was alsoevaluated against large (225 mm³) established L2987 tumors (FIG. 49).The administration of the ChiBR96-ADM conjugate (MR=6.85) at a dose of10 mg/kg equivalent to >10 LCK and 70% cures and 30% partial tumorregressions were observed.

[0717] The antitumor activity of unconjugated ChiBR96 antibody wasevaluated using established (50-100 mm³) L2987 human lung tumorxenografts. As shown in Table 10, ChiBR96 antibody administered at dosesof 100, 200 or 400 mg/kg was not active against established L2987tumors. The antitumor activity of mixtures of ChiBR96 and adriamycin wasnot different from that of adriamycin administered alone. Therefore, theantitumor activity of the ChiBR96-ADM conjugates reflects the efficacyof the conjugate itself rather than a synergistic antitumor effect ofantibody and adriamycin. TABLE 16 Antitumor Activity of Adriamycin,ChiBR96, and Mixtures of ChiBR96 and Adriamycin Against EstablishedL2987 Human Lung Tumor Xenografts Dose (mg/kg)^(a) % Tumor RegressionsTreatment ADM ChiBR96 LCK PR CR Cure No. of Mice Adriamycin 8 — 1.5 0 00 9 ChiBR96 — 400 0 0 0 0 8 — 200 0 0 0 0 8 — 100 0 0 0 0 8 Adriamycin +ChiBR96 8 400 1.8 11 0 0 9 8 200 1.6 0 0 0 9 8 100 1.9 0 0 0 8

[0718] In summary ChiBR96-ADM conjugates demonstrated antigen-specificantitumor activity when evaluated against established L2987 human lungtumors. The antitumor activity of ChiBR96-ADM conjugates was superior tothat of optimized adriamycin, mixtures of ChiBR96 and adriamycin, andequivalent doses of non-binding conjugates. The ChiBR96-ADM conjugateswere approximately 6 fold more potent than unconjugated adriamycin.Cures or complete regressions of established tumors were observed in 50%of animals treated with doses of ≧2.5 mg/kg of ChiBR96-ADM conjugate.

[0719] As shown in FIG. 60, ChiBR96-ADM conjugates (MR—7.88)demonstrated antigen-specific antitumor activity against established(75-125 mm³) MCF7 tumors. The activity of ChiBR96-ADM conjugateadministered at a dose of 5 mg/kg by either the ip or iv route (4.2 LCK)was superior to that of optimized adriamycin (1.4 LCK) or equivalentdoses of non-binding IgG conjugate (1.2 LCK). The antitumor activity ofChiBR96-ADM and non-binding IgG-ADM conjugates is summarize in Table 17.The MTD of ChiBR96-ADM conjugates like that of free adriamycin is lowerin the MCF7 model due to the estradiol supplementation required fortumor growth.

[0720] The antigen-specific antitumor activity and dose response ofChiBR96-ADM conjugates was also evaluated in the RCA human coloncarcinoma model. RCA tumors are less sensitive to unconjugatedadriamycin than are L2987 and MCF7 tumors. In addition, as describedpreviously, RCA tumors have a longer tumor volume doubling time thanL2987 or MCF7 tumors, are more poorly vascularized, and the localizationof radiolabelled BR64 antibody is lower in RCA tumors than in L2987tumors. As Shown in FIG. 61, the antitumor activity of the ChiBR96-ADMconjugate (MR=7.88) administered at a dose of 10 mg/kg was superior tothat of adriamycin and an equivalent dose of non-binding IgG conjugate(MR—7.16). As shown in table 18, the ChiBR96-ADM conjugate tested at adose of 10 mg/kg produced antitumor activity equivalent to >3 LCK. Atthis dose of ChiBR96-ADM conjugate, 80% cures and 11% partial tumorregressions occurred. In this experiment, unconjugated adriamycin showedantitumor activity, equivalent to 0.4 LCK. Thus, in this experiment, theBR96-ADM conjugate produced 89% cures of established tumors whereasunconjugated adriamycin was inactive. TABLE 18 Summary of AntitumorActivity of ChiBR96-ADM Thioether Conjugates Evaluated AgainstEstablished RCA Human Colon Tumor Xenografts Dose (mg/kg)^(a) % TumorRegressions Conjugate ADM ChiBR96 Route LCK PR CR Cure No. of MiceChiBR96-ADM-7.88 10 350 ip >3 11 0 89 9 5 175 ip 0.6 11 22 11 9 2.5 85ip 0.2 0 0 0 9 IgG-ADM-7.16 2.5 85 iv 0.6 11 0 0 9 Adriamycin 10 405 ip0 0 0 0 9 8 0 iv 0.4 0 0 0 9

[0721] In summary, the ChiBR96-ADM conjugate demonstratedantigen-specific antitumor activity in the RCA human colon tumor model.Cures and complete regressions of established RCA tumors were observedfollowing the administration of ChiBR96-ADM conjugate at doses of 5-10mg/kg.

[0722] The invention has been described with reference to specificexamples, materials and data. As one skilled in the art will appreciate,alternate means for using or preparing the various aspects of theinvention may be available. Such alternate means are to be construed asincluded within the intent and spirit of the present invention asdefined by the following claims.

1 4 33 base pairs nucleic acid single linear DNA (genomic) NO NO Plasmidp - #BR96 1 GCTAGACATA TGGAGGTGCA GCTGGTGGAG TCT 33 33 base pairsnucleic acid single linear DNA (genomic) NO NO Plasmid p - #BR96 2GCTGTGGAGA CTGGCCTGGT TTCTGCAGGT ACC 33 720 base pairs nucleic acidsingle linear DNA (genomic) NO NO Mus musculus CDS 1..720 3 ATG GAG GTGCAG CTG GTG GAG TCT GGG GGA GGC TTA GTG CAG CCT GGG 48 Met Glu Val GlnLeu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 TCC CTG AAAGTC TCC TGT GTA ACC TCT GGA TTC ACT TTC AGT GAC TAT 96 Ser Leu Lys ValSer Cys Val Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 TAC ATG TGG GTTCGC CAG ACT CCA GAG AAG AGG CTG GAG TGG GTC GCA 144 Tyr Met Trp Val ArgGln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala 35 40 45 TAC ATT AGT CAA GGTGAT ATA ACC GAC TAT CCA GAC ACT GTA AAG GGT 192 Tyr Ile Ser Gln Gly AspIle Thr Asp Tyr Pro Asp Thr Val Lys Gly 50 55 60 CGA TTC ACC ATC TCC AGAGAC AAT AAG AAC ACC CTG TAC CTG CAA ATG 240 Arg Phe Thr Ile Ser Arg AspAsn Lys Asn Thr Leu Tyr Leu Gln Met 65 70 75 80 AGC CGT CTG AAG TCT GAGGAC ACA GCC ATG TAT TGT GCA AGA GGC CTG 288 Ser Arg Leu Lys Ser Glu AspThr Ala Met Tyr Cys Ala Arg Gly Leu 85 90 95 GAC GAC GGG GCC TGG TTT GCTTAC TGG GGC CAA GGG ACC ACG ACC GTC 336 Asp Asp Gly Ala Trp Phe Ala TyrTrp Gly Gln Gly Thr Thr Thr Val 100 105 110 TCC TCA GGA TCC GGA GGT GGAGGT TCT GGT GGA GGT GGA TCT GGA GGT 384 Ser Ser Gly Ser Gly Gly Gly GlySer Gly Gly Gly Gly Ser Gly Gly 115 120 125 GGA TCT AAG CTT GAT GTT TTGATG ACC CAA ATT CCA GTC TCC CTG CCT 432 Gly Ser Lys Leu Asp Val Leu MetThr Gln Ile Pro Val Ser Leu Pro 130 135 140 GTC AGT CTT GGA CAA GCG TCCATC TCT TGC AGA TCT AGT CAG ATC ATT 480 Val Ser Leu Gly Gln Ala Ser IleSer Cys Arg Ser Ser Gln Ile Ile 145 150 155 160 GTA CAT AAT AAT GGC AACACC TTA GAA TGG TAC CTG CAG AAA CCA GGC 528 Val His Asn Asn Gly Asn ThrLeu Glu Trp Tyr Leu Gln Lys Pro Gly 165 170 175 CAG TCT CCA CAG CTC CTGATC TAC AAA GTT AAC CGA TTT TCT GGG GTC 576 Gln Ser Pro Gln Leu Leu IleTyr Lys Val Asn Arg Phe Ser Gly Val 180 185 190 CCA GAC AGG TTC AGC GGCAGT GGA TCA GGG ACA GAT TTC CTC AAG ATC 624 Pro Asp Arg Phe Ser Gly SerGly Ser Gly Thr Asp Phe Leu Lys Ile 195 200 205 AGC AGA GTG GAG GCT GAGGAT CTG GGA GTT TAT TAC TGC TTT CAA GTT 672 Ser Arg Val Glu Ala Glu AspLeu Gly Val Tyr Tyr Cys Phe Gln Val 210 215 220 CAT GTT CCA TTC ACG TTCGGC TCG GGG ACC AAG CTG GAG ATC AAA CGC 720 His Val Pro Phe Thr Phe GlySer Gly Thr Lys Leu Glu Ile Lys Arg 225 230 235 240 240 amino acidsamino acid linear protein 4 Met Glu Val Gln Leu Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly 1 5 10 15 Ser Leu Lys Val Ser Cys Val Thr Ser GlyPhe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Trp Val Arg Gln Thr Pro Glu LysArg Leu Glu Trp Val Ala 35 40 45 Tyr Ile Ser Gln Gly Asp Ile Thr Asp TyrPro Asp Thr Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Lys AsnThr Leu Tyr Leu Gln Met 65 70 75 80 Ser Arg Leu Lys Ser Glu Asp Thr AlaMet Tyr Cys Ala Arg Gly Leu 85 90 95 Asp Asp Gly Ala Trp Phe Ala Tyr TrpGly Gln Gly Thr Thr Thr Val 100 105 110 Ser Ser Gly Ser Gly Gly Gly GlySer Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Ser Lys Leu Asp Val LeuMet Thr Gln Ile Pro Val Ser Leu Pro 130 135 140 Val Ser Leu Gly Gln AlaSer Ile Ser Cys Arg Ser Ser Gln Ile Ile 145 150 155 160 Val His Asn AsnGly Asn Thr Leu Glu Trp Tyr Leu Gln Lys Pro Gly 165 170 175 Gln Ser ProGln Leu Leu Ile Tyr Lys Val Asn Arg Phe Ser Gly Val 180 185 190 Pro AspArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Leu Lys Ile 195 200 205 SerArg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Val 210 215 220His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 225 230235 240

What is claimed is:
 1. A monoclonal antibody BR96 produced by hybridomaATCC HB 10036, or fragments thereof, and functional equivalents thereofhaving an antigen-binding region that competitively inhibits theimmunospecific binding of monoclonal antibody BR96, having specificimmunological reactivity with human carcinoma cells, said antibodycharacterized by being capable of internalizing within the carcinomacells with which it reacts, mediating antibody-dependent cellularcytotoxicity and complement-dependent cytotoxicity activity, and/orkilling of said human carcinoma cells in the absence of host effectorcells or complement.
 2. Hybridoma HB 10036 as deposited with the ATCC.3. An Fab, F(ab′)₂ or Fv fragment of the antibody of claim
 1. 4. Animmunoconjugate comprising a molecule containing the antigen-bindingregion of the BR96 monoclonal antibody joined to a cytotoxic agent. 5.The immunoconjugate of claim 4, wherein the molecule comprises BR96monoclonal antibody or fragments thereof.
 6. The immunoconjugate ofclaim 4, wherein the molecule comprises chimeric human/murine BR96antibody or fragments thereof.
 7. The immunoconjugate of claim 5,wherein the fragments are selected from the group consisting of Fv,F(ab′) and F(ab′)₂ fragments.
 8. The immunoconjugate of claim 6, whereinthe fragments are selected from the group consisting of Fv, F(ab)′ andF(ab′)₂ fragments.
 9. A method for selectively killing tumor cellsexpressing the antigen that immunospecifically binds to BR96 monoclonalantibody comprising reacting the immunoconjugate of claim 4 with saidtumor cells.
 10. A recombinant single-chain immunotoxin moleculecomprising a cloned heavy chain Fv portion and a cloned light chain Fvportion of the BR96 monoclonal antibody joined to a cytotoxic agent. 11.A bispecific antibody with a binding specificity for two differentantigens, one of the antigens being that with which the monoclonalantibody of claim 1 binds.
 12. The bispecific antibody of claim 11,wherein one of the antigens comprises a variant of Le^(y) determinantwhich includes an epitopic site containing fucose α1-3.
 13. A monoclonalantibody, the antigen-binding region of which competitively inhibits theimmunospecific binding of monoclonal antibody BR96 produced by hybridomaHB 10036 to its target antigen.
 14. The antibody of claim 13, whereinsaid antigen comprises a variant of Le^(y) determinant which includes anepitopic site containing fucose α1-3.
 15. A human/murine recombinantantibody, the antigen-binding region of which competitively inhibits theimmunospecific binding of monoclonal antibody BR96 produced by hybridomaHB 10036 to its target antigen.
 16. The antibody of claim 1, wherein theantibody is conjugated to a therapeutic agent to form an antibodyconjugate.
 17. The antibody of claim 16, wherein the therapeutic agentis an anti-tumor drug, a cytotoxin, a radioactive agent, a secondantibody or an enzyme.
 18. The antibody of claim 17, wherein thecytotoxin is a ribosome binding toxin.
 19. The antibody of claim 18,wherein the ribosome binding toxin is ricin A.
 20. A compositioncomprising a combination of an immunoconjugate comprising the antibodyof claim 1 linked to an enzyme capable of converting a prodrug into acytotoxic drug, and said prodrug.
 21. A pharmaceutical compositionuseful in the treatment of carcinomas comprising a pharmaceuticallyeffective amount of the antibody of claim 1 and an acceptable carrier.22. A pharmaceutical composition useful in the treatment of carcinomascomprising a pharmaceutically effective amount of at least one antibodyconjugate according to claim 16 and an acceptable carrier.
 23. A methodof treating carcinomas in vivo comprising administering to a patient apharmaceutically effective amount of a composition containing theantibody of claim
 1. 24. A method for determining the presence ofcarcinoma in human tissue comprising contacting a specimen of saidtissue with the antibody of claim 1 and detecting the binding of saidantibody to said tissue.
 25. The method of claim 24, wherein saidantibody is labeled so as to directly or indirectly produce a detectablesignal with a compound selected from the group consisting of aradiolabel, an enzyme, a chromophore and a fluorescer.
 26. A method forimaging carcinoma comprising administering to a patient intravenouslythe antibody of claim 1 in an amount effective for detection of thecarcinoma, allowing the antibody to bind to carcinoma cells and tolocalize to the site of carcinoma cells and detecting said antibodybound to the carcinoma cells.
 27. The method of claim 26, wherein saidantibody is labeled so as to directly or indirectly produce a detectablesignal with a label selected from the group consisting of a radiolabel,an enzyme, a chromophore, and a fluorescer.
 28. A monoclonalanti-idiotypic antibody reactive with an idiotope on the antibody ofclaim
 1. 29. A diagnostic kit comprising: a) the antibody of claim 1;and b) a conjugate of a detectable label and a specific binding partnerof the antibody of (a) above.
 30. The diagnostic kit of claim 29,wherein the label is selected from the group consisting of enzymes,radiolabels, chromophores and fluorescers.
 31. The immunoconjugate ofclaim 4, wherein the cytotoxic agent is selected from a group consistingof antimetabolites, alkylating agents, anthracyclines, antibiotics,anti-mitotic agents, and chemotherapeutic agents.
 32. Theimmunoconjugate of claim 4, wherein the cytotoxic agent is selected froma group consisting of ricin, doxorubicin, daunorubicin, taxol, ethidiumbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, dihydroxy anthracin dione, actinomycin D,1-dehydrotestosterone, and glucocorticoid.
 33. A method for curing asubject suffering from a cancer, the cancer being characterized as agroup of cells having a tumor associated antigen on the cell surface,which method comprises administering to the subject a cancer killingamount of a tumor targeted antibody joined to a cytotoxic agent underconditions which permit the antibody so joined to bind the tumorassociated antigen on the cell surface so as to kill the cells so boundthereby curing the subject.
 34. The method of claim 33, wherein thetumor targeted antibody is an internalizing antibody.
 35. The method ofclaim 33, wherein the tumor targeted antibody is an internalizingantibody which recognizes and binds to a Le^(y) determinant.
 36. Amethod of inhibiting the proliferation of mammalian tumor cells whichcomprises contacting the mammalian tumor cells with a proliferationinhibiting amount of a tumor targeted antibody joined to doxorubicin soas to inhibit proliferation of the mammalian tumor cells.
 37. The methodof claim 36, wherein the tumor targeted antibody is the monoclonalantibody BR96 produced by hybridoma ATCC HB
 10036. 38. The method claim36, wherein the tumor targeted antibody is a chimeric antibody ChiBR96produced by the hybridoma having the identifying characteristics of HB10460 as deposited with the ATCC.
 39. The method of claim 36, whereinthe tumor targeted antibody is the bispecific antibody with a bindingspecificity for two different antigens, one of the antigens being thatwith which the monoclonal antibody BR96 produced by hybridoma ATCCHB10036 binds.
 40. The method of claim 36, wherein the tumor targetedantibody is the monoclonal antibody, the antigen-binding region of whichcompetitively inhibits the immunospecific binding of monoclonal antibodyBR96 produced by hybridoma HB 10036 to its target antigen.
 41. Themethod of claim 36, wherein the tumor targeted antibody is thehuman/murine recombinant antibody, the antigen-binding region of whichcompetitively inhibits the immunospecific binding of monoclonal antibodyBR96 produced by hybridoma HB 10036 to its target antigen.
 42. A methodfor selectively killing tumor cells expressing the antigen thatimmunospecifically binds to BR96 monoclonal antibody comprising reactingan immunoconjugate comprising a molecule containing the antigen-bindingregion of the BR96 monoclonal antibody joined to doxorubicin with thetumor cells so as to obtain a BR96/doxorubicin-tumor cell complexthereby permitting the doxorubicin to kill the tumor cells so complexed.43. A method of inhibiting the proliferation of mammalian tumor cellswhich comprises contacting the mammalian tumor cells with a sufficientconcentration of an immunoconjugate comprising a molecule containing theantigen-binding region of the BR96 monoclonal antibody joined todoxorubicin so as to obtain a BR96/doxorubicin-tumor cell complexthereby inhibiting proliferation of the mammalian tumor cells socomplexed.
 44. A method for treating a subject suffering from aproliferative type disease characterized by cells having the BR96antigen on the cell surface which comprises administering to the subjectan effective amount of an immunoconjugate comprising the antigen-bindingregion of the BR96 monoclonal antibody joined to doxorubicin such thatthe immunoconjugate binds the BR96 antigen and kills said cells therebytreating the subject.